BR112014015077A2 - method for producing a di- or trifunctional alkane - Google Patents

Abstract

  METHOD TO PRODUCE A DI- OR TRIFUNCTIONAL ALKAN. Embodiments of the present invention pertain to methods for the biosynthesis of di- or tri-functional C7 alkanes in the presence of isolated enzymes or in the presence of a recombinant host cell expressing those enzymes. The di- or trifunctional C7 alkanes are useful as production intermediates and nylon-7, nylon-7, x, nylon-x, 7, and polyesters.

Description

“METHOD TO PRODUCE A DI- OR TRIFUNCTIONAL ALKAN” Field of the invention

[001] Embodiments of the present invention relate to methods for the biosynthesis of di- or trifunctional C7 alkanes in the presence of isolated enzymes or in the presence of a recombinant host cell expressing those enzymes. The di- or trifunctional C7 alkanes are useful as production intermediates and nylon-7, nylon-7, x, nylon-x, 7, and polyesters. Background of the invention

[002] The promotion of sustainable practices by the chemical industry is focused on reducing energy use, recycling raw materials, and reducing by-products produced during the production of goods. However, the chemical industry depends, vitally, on oil and natural gas as a basic raw material. Given the state of the associated optimization with the largest petrochemical processes, a significant change in the use and structuring of raw material production will require a reduction in energy, emissions and costs below its current level. Biotechnology offers a new approach to the production of intermediates by the chemical industry and final products from renewable carbohydrates, such as sugars derived from plant, or other renewable raw material, such as fatty acids. In addition, biotechnology also offers a way to use low value waste or streams, such as glycerol from production and biodiesel, or to transform raw materials derived from petrochemicals, such as benzene, toluene, and polycyclic aromatic hydrocarbons (PAHs) into products useful in bioprocesses that are more benign to the environment than existing chemical processes.

[003] The publications WO 2010/0203600 and WO 2011/031147 are incorporated by reference in their entirety. There is a need in the art for cost-effective biosynthetic routes for chemical intermediates for nylon-7, nylon-7, Xx, nylon-x, 7, and polyesters and the final products of nylon-7, nylon-7, x , nylon-x, 7, and polyesters especially from a variety of renewable and non-renewable raw materials. Summary of the invention

[004] This description is based at least in part on the development - * of enzyme systems and recombinant hosts for biosynthesis of di- or trifunctional c7 alkanes that are useful intermediates (monomers) for the production of nylon-7, nylon-7, x, nylon-x, 7, and polyesters. In particular, as described here, intermediates useful in the production of nvylon-7, nvylon-7, x, nvylon-x, 7, and polyesters can be bio-synthetically produced from renewable raw materials, such as plant-derived sugars and glycerol, or from raw materials derived from petrochemicals such as benzene or cyclohexane carboxylates. In some embodiments, the production of di- or trifunctional C7 alkanes in the end proceeds through a common intermediate, pyelic acid semialdehyde (also known as 7-oxoheptanoate), although there are a number of different raw materials that can be used and a number of ways to produce pyelic acid semialdehyde. Figure 1 is a schematic view representing the bio-transformation of semialdehyde and pyelic acid into various difunctional C7 alkanes.

[005] For example, pyelic acid semialdehyde can be derived from pimeloyl-coenzyme A (CoA), pimeloyl- [acp- (acp)] carrier protein, pyelic acid (also known as heptane 1,7-dioate), or suberate da-keto. As described here, pimeloil-CoA, pimeloil- [acp] l, and pyelic acid semialdehyde can be derived from a number of sources, including from a number other than the naturally occurring metabolic pathway that forms pimeloyl-CoA, pimeloyl- [acp] or pyelic acid semialdehyde as an intermediate in the naturally occurring metabolic route.

[006] In addition, pimeloyl-CoA and pyelic acid can also be derived from the corresponding 2-heptane-dioic acid enoate or its corresponding CoA esters, 2-7 heptanedioyl-CoA. In some embodiments, these C7 2-enoates are derived from D, L-diaminopimelate or 2-oxopimelate. In some embodiments, the production of difunctional C7 alkanes (for example, 7-amino-heptanoic acid) proceeds via suberate α-keto or α-amino suberate, which can be derived from D, L-diaminopimelate. In other embodiments, Q & - keto suberate or a-amino suberate is derived from a-ketopimelate.

[007] More specifically, this document provides a variety of methods for converting compounds to produce intermediates useful for the manufacture of nylon-7, nylon-7.7 and polyesters. It is not required that all possible steps of either method be performed and any of the methods can be completed when a desired compound is obtained. Thus, any method can be terminated after the production of, for example, pyelic acid, 7-amino-heptanoic acid, enantolactam, 1,7-diamino-heptane, or 7T-hydroxy-hepanic acid.

[008] A first method of converting a compound may include contacting a compound selected from 2,6-

diaminopimelate (2,6-DAP) and ar-amino-pimelate (AAP) with an enzyme that catalyzes the reductive deamination of 2,6-DAP in 6-amino-2-heptenedioic acid (6A2HA) or the reductive deamination of AAP in 2-heptene dioic acid (2 HDA), so that 6A2HA or 2 HDA is produced.

[009] The compound can be 2,6-DAP and the first method can also involve contacting 6A2HA with an enzyme that catalyzes the reduction of 6A2HA enoate in AAP, so that AAP is produced.

[0010] AAP can be contacted with an enzyme that catalyzes the reductive deamination of AAP in 2HDA, just as 2 HDA is produced.

[0011] 2HDA can be contacted with an enzyme that catalyzes the enoate reduction of 2HDA to pyelic acid (PA), so that PA is produced.

[0012] PA can be contacted with an enzyme that catalyzes the reduction of PA carboxylic acid to pyelic acid semialdehyde (PAS), so that PAS is produced.

[0013] PAS can be contacted with an enzyme that catalyzes the amination of PAS semialdehyde in 7-amino-heptanoic acid (7AHA), so that 7AHA is produced.

[0014] 7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA into enantolactam (ENTL), so that ENTL is produced.

[0015] A second method of converting the compound may include, after performing all the steps of the first method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of 7AHA aldehyde into 7-amino-heptanal (7 AHT ) so that AHT is produced.

[0016] 7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group in 7AHT to produce 1,7-diaminoheptane (1,7-DAH), so that 1,7-DAH is produced.

[0017] A third method of converting the compound may include, after performing all the steps of the first method until production and 2 HDA, contacting 2HDA with an enzyme that catalyzes the transfer of coenzyme A (CoA) in 2HDA to produce 2-heptene-CoA diacid (2 HDA-CoA), so that 2 HDA-CoA is produced.

[0018] 2 HDA-CoOA can be contacted with an enzyme that catalyzes the enoate reduction of 2 HDA-CoA in pimeloyl-CoA (PCoA), so that PCoA is produced.

[0019] PCoA can be contacted with an enzyme that catalyzes the hydrolysis of the PCoA thioester in PA, so that PA is produced.

[0020] PA can be contacted with an enzyme that catalyzes the reduction and carboxylic acid of PA in PAS, so that PAS is produced.

[0021] PAS can be contacted with an enzyme that catalyzes the amination and semialdehyde of PAS in 7AHA, so that 7AHA is produced.

[0022] 7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0023] A fourth method of converting a compound may include, after performing all the steps of the third method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of the 7AHA aldehyde into 7AHT, so that 7AHT is produced.

[0024] 7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1.7 DAH, so that 1,7-DAH is produced.

[0025] A fifth method of converting a compound may include, after performing all steps from the first method to production and AAP, contacting the AAP with an enzyme that catalyzes the transfer of an amino group to the AAP to produce the -keto-pimelate (AKP), so that AKP is produced.

[0026] AKP can be contacted with one or more enzymes that catalyze the reduction of AKP acetone in a-hydroxy-pimelate (AHP), so that AHP is produced.

[0027] AHP can be contacted with an enzyme that catalyzes the transfer of CoA into AHP to produce Qar-hydroxypimelate-CoA (AHP-CoA), so that AHP-CoA is produced.

[0028] AHP-CoA can be contacted with an enzyme that catalyzes the dehydration of AHP-CoA in 2HDA-CoA, so that 2Z2HDA-CoA is produced.

[0029] 2HDA-CoA can be contacted with an enzyme that catalyzes the reduction and enoate of 2HDA-CoA in PCoA, so that PCoA is produced.

[0030] PCoA can be contacted with an enzyme that catalyzes the thioester hydrolysis of PCoA in PA, so that PA is produced.

[0031] PA can be contacted with an enzyme that catalyzes the reduction and carboxylic acid of PA in PAS, so that PAS is produced.

[0032] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7AHA, so that 7AHA is produced.

[0033] T7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, where ENTL is produced.

[0034] A sixth method of converting a compound may include, after performing all the steps of the fifth method until production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation and aldehyde of 7AHA into 7AHT, so that 7AHT be produced.

[0035] 7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group in 7AHT to produce 1.7DAH, so that 1,7-DAH is produced.

[0036] A seventh method of converting a compound can include, after performing all the steps of the fifth method until the production of AHP, contacting AHP with an enzyme that catalyzes the dehydration of AHP in 2HDA, so that 2HDA is produced .

[0037] 2HDA can be contacted with an enzyme that catalyzes the reduction of 2HDA enoate in pyelic acid (PA), so that PA is produced.

[0038] PA can be contacted with an enzyme that catalyzes the reduction and carboxylic acid of PA in PAS, so that PAS is produced.

[0039] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7AHA, so that 7AHA is produced.

[0040] 7AHA can be contacted with an enzyme that catalyzes HDA amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0041] An eighth method of converting a compound may include, after performing all the steps of the fifth method until production and 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of 7AHA aldehyde into 7AHT, so that 7 AHT is produced.

[0042] 7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group in 7AHT to produce 1.7 DAH, so that 1,7-DAH is produced.

[0043] A ninth method of converting a compound may include, after performing all the steps of the seventh method until the production of 2HDA, contacting 2HDA with an enzyme that catalyzes the transfer of CoA into 2 HDA to produce 2HDA-CoA , so that 2HDA-CoA is produced.

[0044] 2HDA-CoA can be contacted with an enzyme that catalyzes the reduction of 2HAD-CoA enoate in PCoa, so that PCoA is produced.

[0045] PCOoA can be contacted with an enzyme that catalyzes the hydrolysis of PCoA thioester in PA, so that PA is produced.

[0046] PA can be contacted with an enzyme that catalyzes the reduction of PA carboxylic acid in PAS, so that PAS is produced.

[0047] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7 AHA, so that 7AHA is produced.

[0048] 7 AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0049] A tenth method of converting a compound can convert a compound can include, after completion and all the steps of the ninth method up to the production of 7 AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of the 7AHA aldehyde into 7AHT, so that 7AHT is produced.

[0050] T7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1.7DAH, so that 1,7-DAH is produced.

[0051] An eleventh method of converting a compound may include, after performing all the steps of the fifth method until the production of AKP, contacting the AKP with an enzyme that catalyzes the lengthening of the AKP acid chain in a-keto-suberate (AKS), so that AKS is produced.

[0052] AKS can be contacted with an enzyme that catalyzes the transfer of an amino group to AKS to produce a-amino suberate (AAS), so that AAS is produced. AAS can be contacted with an enzyme that catalyzes the decarboxylation of AAS amino acid in 7AHA, so that 7AHA is produced.

[0053] 7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0054] A twelfth method of converting a compound may include, after performing all the steps of the eleventh method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of aldehyde from 7AHA to 7AHT, from so that 7 AHT is produced.

[0055] 7AHT can be contacted with an enzyme that catalyzes the transfer of a group to 7AHT to produce 1,7-DAH, so that 1,7-DAH is produced.

[0056] A thirteenth method of converting a compound may include after performing all steps of the eleventh method until the production of AKS, contacting AKS with an enzyme that catalyzes the acid decarboxylation of AKS keto in PAS , so that PAS is produced.

[0057] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7AHA, so that 7AHA is produced.

[0058] 7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0059] A fourteenth method of converting a compound can include after performing all the steps of the thirteenth method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of aldehyde from 7AHA to 7AHT, so that 7 AHT is produced.

[0060] T7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1.7 DAH, so that 1,7-DAH is produced.

[0061] A fifteenth method of converting a compound can include, after performing all the steps of the fifth method until the production of PCoA, contacting the PCOA with an enzyme that catalyzes the reduction of PCoA to PAS, so that PAS is produced.

[0062] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS to 7AHA, so that 7AHA is produced.

[0063] 7AHA can be contacted with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0064] A sixteenth method of converting a compound may include, after performing all steps of the fifteenth method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of aldehyde from 7AHA to 7AHT, from so that 7 AHT is produced.

[0065] T7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group in 7AHT to produce 1.7DAH, so that 1,7-DAH is produced.

[0066] A seventeenth method of converting a compound may include, after performing all the steps of the ninth method until the production of PCoA, contacting the PCOA with an enzyme that catalyzes the reduction of PCoA in PAS, so that PAS is produced.

[0067] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7AHA, so that 7AHA is produced.

[0068] 7AHA can be counted on an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0069] An eighteenth method of converting a compound can include after performing all steps of the seventeenth method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of aldehyde from 7AHA to 7AHT, so that 7 AHT is produced.

[0070] 7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1.7DAH, so that 1,7-DAH is produced.

[0071] A nineteenth method of converting a compound may include contacting a selected compound of 6A2HA and 2HAD with an enzyme that catalyzes the reduction of 2HDA enoate in PA, so that AAP or PA is produced. It should be understood that, after production and AAP or PA, any of the steps after the production of AAP or PA described above from the first to the eighteenth method can be performed.

[0072] A twentieth method of converting a compound may include contacting a selected compound of PCoA and pimeloyl Lacp] (PACP) with an enzyme that catalyzes the hydrolysis of PCOA or PACP thioesterase into PA, where PA is produced.

[0073] PA can be contacted with an enzyme that catalyzes the reduction of PA carboxylic acid in PAS, so that PAS is produced.

[0074] PAS can be contacted with an enzyme that catalyzes the semialdehyde amination of PAS in 7AHA, so that 7AHA is produced.

[0075] 7AHA with an enzyme that catalyzes the amide hydrolysis of 7AHA in ENTL, so that ENTL is produced.

[0076] A twenty-first method of converting a compound may include, after performing all the steps of the twenty-first method until the production of 7AHA, contacting 7AHA with an enzyme that catalyzes the dehydrogenation of aldehyde from 7AHA to 7AHA, so that 7AHT is produced.

[0077] T7AHT can be contacted with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1.7DAH, so that 1,7-DAH is produced.

[0078] A twenty-second method of converting a compound may include contacting AKP with one or more enzymes that catalyze AKP reduction and acetone in AHP, so that AHP is produced. AKP may have been produced by elongating QO-keto adipate or Qa-keto-glutarate. AHP can then be converted to PA or PCoA. It should be understood that, after production and PA or PCoA, any of the steps after the production of PA or PCoA described above in the first to the eighteenth method can be performed.

[0079] A twenty-third method of converting a compound may include converting AKP to AKS in an a-keto acid chain elongation. It should be understood that, after the production of AKS, any of the steps after the production of AKS described above in the first to the eighteenth method can be performed.

[0080] A twenty-fourth method of converting a compound may include contacting PAS with an enzyme that catalyzes the amination and semialdehyde and PAS in 7AHA, so that 7AHA is produced. PAS may have been obtained by converting 2.6 DAP, AKG, PCoA, or PACP. It should be understood that, after the production of AHA, any of the steps after the production of AHA described above in the first to the eighteenth method can be performed.

[0081] A twenty-fifth method of converting a compound may include contacting a compound selected from PCoA and PACP with an enzyme that catalyzes the reduction of the compound to PAS, so that PAS is produced. It should be understood that, after production and PAS, any of the steps after production and PAS described above in the first to eighteenth method can be performed. In addition, any of the steps described in the first and eighteenth method and in the twenty-second method leading to the production of PCoA can be performed before the twenty-fifth method.

[0082] A twenty-sixth method of converting a compound may include contacting PAS with an enzyme that catalyzes the dehydrogenation of PAS alcohol to 7-hydroxyheptanoic acid (7HHA), so that 7HHA is produced. It should be understood that any of the steps described in the first to the eighteenth method and in the twenty-second method, twenty-second, twenty-third, and twenty-fifth methods leading to the production of PAS can be performed before the twenty-sixth method.

[0083] In any of the described methods, the enzymes can be used in purified form. Alternatively, the enzyme (s) can be used in the form of a cell lysate or a partially purified cell lysate. In addition, the enzyme (s) can be in a cell expressing recombinantly he / they.

[0084] In the methods described, an enzyme that catalyzes reductive deamination may include an ammonia lyase. Ammonia lyase may be in EC 4.3.1, for example, EC 4.3.1.1; EC

4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC

4., 3.1.14; EC 4.3.1.23 or EC 4.3.1.24.

[0085] In the described methods, an enzyme that catalyzes the reduction and 2-enoate can include an enoate reductase. Enoate reductase can be in EC 1.3.1, for example, EC

1.3.1.8; EC 1.3.1.9; EC 1.3.1.10, such that the FabI gene product; Ec 1.3.1.31; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 so that the ester or tdter gene product (Nishimaki et al., “J. Biochem.”, 1984, 95, 1315 - 1321; Shen et al., “Appl Environ. Microbiol.”, 2011 , 77 (9), 2905 - 2915; Bond Watts et al., "Biochemistry", 2012, 51, 6827 - 6837); or pimeloyl-CoA dehydrogenase in EC 1.3.1.62 so that PimC / PimD or ThnJ / ThnK. Enoate reductase may be in EC

1.3, such as EC 1.3.8.1; EC 1.3.99.3; EC 1.3.99.B10 or Syntrophus aciditrophicus 2,3-didehydropyelyl-CoA reductase and homologues thereof. Enoate reductase can act on trans-2-enoyl-dicarboxylic acids, or thioesters of dicarboxylic acids such as 2-heptane dioic acid-CoA (trans-2,3-didehydropylyl1-CoA) or 2-heptene dioic acid-

[acp] (trans-2,3-didehydropylyl- [acp]).

[0086] In the methods described, an enzyme that catalyzes the reduction of carboxylic acid can include a carboxylic acid reductase. The carboxylic acid reductase can be EC 1.2.99, for example, EC 1.2.99.6. The carboxylic acid reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenase, such as ThnG in Shphingomonas and Cupriavidus spp.

[0087] In the described methods, an enzyme that catalyzes the amination and semialdehyde can include an O-transaminase. Q-transaminase can be EC 2.6.1, for example, EC 2.6.1.18; EC

2.6.1.19; EC 2.6.1.11; EC 2.6.1.39; EC 2.6.1.1.48; or EC

2.6.1.62.

[0088] In the methods described, an enzyme that catalyzes the hydrolysis of the CoA-thioester thioester can include a thioesterease, an acid-thiol ligase, or a CoA transferase. Thioesterase can be in EC. 3.1.12, for example, such as the YciA, tesB or AcotI3 gene product; EC 3.1.2.2; EC

3.1.2.18; EC 3.1.2.19; or EC 3.1.2.20. The acid-thiol ligase / CoA synthetase can be in EC 6.2.1, for example, EC

6.2.1.3; EC 6.2.15; EC 6.2.1.14; or EC 6.2.1.23. CoA transferase can be in EC 2.8.3, for example, EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14, or the ThnH gene product that catalyzes the reversible transfer of CoA in pyelic acid.

[0089] In the methods described, the enzyme that catalyzes the hydrolysis of the vehicle thioester acyl-protein ([ACP]) thioesters, can include an acyl- [ACP] thioesterase from EC

3.1.2, for example, EC 3.1.2.14 or EC 3.1.2.21, an acyl- [ACP] synthase of EC 6.2.1, for example EC 6.2.1.14, so that BioW; and EC 6.2.1.20.

[0090] In the described methods, an enzyme that catalyzes the transfer of an Or amino group can include an amino transferase acid. The amino transferase acid can be a selective L- or D-amino transfrase in Ec 2.6.1, for example, EC

2.6.1.39; EC 2.6.1.42 or EC 2.6.1.21, or a 2-aminohexanoate amino transferase from EC 2.6.1.67. The enzyme that catalyzes the transfer of an alpha amino group may also be a dehydrogenase of the class EC 1.4.1.-., Such as glutamate dehydrogenase, which depending on the cofactor used belongs to EC 1.4.1.2; EC 1.4.1.3; or EC 1.4.1.4, or a diaminopimelate dehydrogenase from EC 1.4.1.16.

[0091] In the methods described, an enzyme that catalyzes ketone reduction can be a carbonyl reductase in EC

1.1.1.- or EC 1.1.99, -. Carbonyl reductase can include Ec

1.1.1.184; Ec 1.1.1.79; EC 1.1.1.B3; EC 1.1.1.B4 or 2-hydroxyglutaryl dehydrogenases / alpha-ketoglutarate reductases from EC 1.1.99.2 or EC 1.1.99.6.

[0092] In the described methods, an enzyme that catalyzes the decarboxylation of a-keto acid can be a QOa-keto acid decarboxylase in EC 4.1.1, for example, 4.1.1.1; 4.1.7;

4.1.1.72; or an acetolactate synthase in class EC 2.2.1.6 or EC 2.2.2.6.

[0093] In the described methods, the enzyme that catalyzes the Qa-amino acid decarboxylation may be an Oaramino acid decarboxylase of the class EC 4.1.1.- ,. Α-amino acid decarboxylase can include EC 4.1.1.11; EC 4.1.1.15; EC

4.1.16; Ec 4.1.1.18; EC 4.1.120; EC 4.1.1.45; and EC 4.1.1.86.

[0094] In the described methods, an enzyme that catalyzes the reduction and CoAa-ester of a dicarboxylic acid to the corresponding O semialdehyde (from, for example, PCoA in PAS)

it may be a fatty acyl-CoA reductase. The fatty acyl-CoA reductase can be in Ec 1.2.1.-, for example, EC 1.2.1.3; EC

1.2.1.10; EC 1.2.1.22; EC 1.2.1.50; EC 1.2.1.57 and EC

1.2.1.76.

[0095] In the described methods, an enzyme that catalyzes the reduction of [ACP] thioester to the corresponding semialdehyde (from, for example, PACP in PAS) can be a fatty acyl-CoA reductase acting on CoaA-esters and [ACP] esters described “above, or an acyl-acyl-protein reductase vehicle of, for example, EC 1.2.1.80.

[0096] In the described methods, an enzyme that catalyzes the dehydrogenation of the aldehyde to the carboxylic acid can be an aldehyde dehydrogenase or an aldehyde oxidase. The aldehyde dehydrogenase can be at EC 1.21, for example, EC 1.2.13; EC 1.2.14 or EC 1.2.1.63. The aldehyde dehydrogenase / carboxylic acid reductase can also be in EC 1.2.99, for example, EC 1.2.99.6. Carboxylic acid reductase may also belong to NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas & € and Cupriavidus spp. The aldehyde oxidase can be in EC 1.2.3, for example, 1.2.3.1.

[0097] In the described methods, an enzyme that catalyzes the closure of the 7T-aminoheptanoic acid ring for enantolactam can be an amidohydrolase from EC 3.5.2, for example, 3.5.2.11.

[0098] In the methods described, an enzyme that catalyzes dehydration (of, for example, AHP or AHP-CoA) can be a hydro-lyase. The hydro-lysis can be in EC 4.2.1, for example, EC 4.2.1.2; EC 4.2.1.59; EC 4.2.1.61; 4.2.1.17 or 4, .2.1.18.dehydratase can also be 2-hydroxyglutyl-CoA dehydratase described in clostridia and fusobacterium to which an EC number has not yet been assigned, that is, expressed with its activator (HgdCAB) or a 2 -hydroxycil CoA dehydratase from anaerobic bacteria.

[0099] In the described methods, the enzyme that catalyzes the elongation of the QO-keto acid chain can include one of the many sets of enzymes including, AksSA, AksD, AKksSE and AksF. AksA can be in EC 2.3.3, for example, EC 1.1.187. One or more (for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 12 or more, 14 or more , 18 or more, or 20 or more) of the a-keto acid chain lengthening enzymes can be obtained from the methanogenic bacteria.

[00100] In the described methods, an enzyme that catalyzes alcohol dehydrogenation can include an alcohol dehydrogenase. Alcohol dehydrogenase can include EC 1.1.1.1. or EC 1.1.1.2 or l1, .1.1.21. It can also be, for example, Zymomonas mobilis adhA, Zymomonas mobilis adB, butanol dehydrogenase from Clostridium acetobutylicum, Saccharomyces ADHIV, and S. cerevisiae ADH6. In the methods described, the enzyme that catalyzes the dehydrogenation of alcohol can be an alcohol oxidase. Alcohol oxidase can be in class EC 1.1.3.13.

[00101] PCoA or PACP used in the document methods can be derived from acetyl-CoA or benzoyl-Coa or from any metabolic pathway such as biotin biosynthesis, benzoate degradation, cyclohexane carboxylate route, or malonyl- CoA or QM-fatty acid oxidation. Acetyl-CoA can be derived from raw material from a renewable source comprising cellulosic raw material, sugars, glycerol, or fatty acids. In addition, acetyl-CoA can be derived from syngas, methane or methanol. Benzoyl-CoA can be derived from polycyclic aromatic hydrocarbons. In addition, to be derived from PCOA or PACP or PA, PAS can be derived from the tetralin degradation route.

[00102] The 2,6-DAP used in the document's methods can be produced by a lysine-producing organism losing the diaminopimelate decarboxylase. 2.6 DAP is derived from any fermentable carbon source, including sugars, glycerol, fatty acids, synthesis gas, methane or methanol.

[00103] This document features a bioderivative nylon-7, nylon-7, x, nylon-x, 7, and polyester. It also includes nylon-7 produced by a process that includes polymerization 7-AHA, where 7 AHA is derived from PAS or AAS. Also provided by the document is a nylon-7.7 produced by a process that includes polymerization of PA and 1.7 DHA, where PAH is derived from PCoA; PACP; or 2HDA and 1,7DHA is derived from 7AHA. The document also configures nylon-7 produced by a process that includes polymerization 7 AHA made by any of the methods described above that leads to production and 7AHA. Another aspect of the invention is nylon-7.7 produced by a process comprising polymerization 1.7 DHA and PA, where 1.7 DHA and PA is made by any of the methods described above which leads to the production of PA .

[00104] This document also provides a substantially pure culture of host cells, a substantial number of which comprises and expresses one or more exogenous nucleic acids encoding one or more enzymes involved in the biosynthesis of one or more polymeric monomers selected from 7AHA ; PAN; 1.7 DAH; ENTL; and 7HHA. These enzymes include fatty acyl-CoA reductase, acyl- [acp] reductase, thio-ester hydrolase, ammonia-lyase, enoato-reductase, amino acid aminotransferase, CoA-transferases, acid-thiol ligases, hydro-liases, carbonyl reductase , carboxylic acid reductases, O-transaminases, a-keto acid decarboxylases, aldehyde dehydrogenase, deamination dehydrogenase, alcohol dehydrogenases, aldehyde oxidase, alcohol oxidase, amidohydrolases, acetolactate synthase decarboxylation and elongation catalyzing enzymes a-keto acid chain such as AKkSA, AksD, AKksSE and AksF. As used here, a “substantial number” is at least 10% (for example, at least: 20%, 30%; 40%; 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95%, 97%, 98%, 99% or even 100%) of cells in the culture.

[00105] The cells of the culture can be prokaryotic cells, such as, without limitation, the genus Escherichia, such as the species Escherichia coli; the genus Clostridia, such as the species Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; the genus Corynebacteria such as the species Corynebacterium glutamicum; the genus Cupriavidus such as the species Cupriavidus necator or Cupriavidus metallidurans; the genus Pseudomonas such as the species Pseudomonas fluorescens, Pseudomonas - “putida or Pseudomonas oleavorans; the genus Delftia such as the species Delftia acidovorans; the genus Bacillus tal as the species Bacillus subtillis; of the genus Lactobacillus as well as the species lactobacillus delbrueckii; or of the genus lactococcus such as the species Lactococcus lactis.

[00106] Alternatively, the cells in the culture may be eukaryotic cells, for example, fungal cells such as yeast cells. Yeast cells can be from a yeast or a fungus, such as, without limitation, the Aspergillus genus such as the Aspergillus species such as the Aspergillus niger species; the genus Sacharomyces such as the species Saccharomyces cerevisiae; the genus Candida such as the three species C. tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. maltosa, C. parapsilosis, and C. zeylenoides, the genus Pichia as the species Pichia pastoris ; the genus Yarrowia such as the species Yarrowia lypolytica; the genus Issatchenkia such as the species Issathenkia orientalis; the genus Debaryomyces such as the species Debaryomyces hansenii; the genus Arxula such as the species Arxula adenoinivorans; the genus Klyyveromyces such as the species Kluyveromyces lactis; the genus Exophiala, the genus Mucor, the genus Trichoderma, the genus Paecilomyces, the genus Scedosporium, or the genus Ophiostoma.

[00107] The exogenous enzymes that can be expressed by cells are as follows. Ammonia lyases may include ammonia lyases in EC 4.3.1, for example, EC 4.3.1.1; EC

4.3.1.2, EC 4.3.1.3; EC 4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC

4.3.1.14; EC 4.3.1.23 or EC 4.3.1.24. Enoate reductase can include an enoate reductase in EC 1.3.1, for example, EC

1.3.1.3; EC 1.3.1.9; EC 1.3.1.10; EC 1.3.1.31; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 or pimeloyl-CoA dehydrogenase in EC

1.3.1.62 such as PimC / PimD or ThnJ / ThnK. Enoate reductase can also be in EC 1.3., Such as EC 1.3.8.1 or EC

1.3.99.3; EC 1.3.99.B10 or Syntrophys aciditrophicus 2,3- didehydropyelil-CoA reductase and homologues thereof. Carboxylic acid reductases include a carboxylic acid reductase in EC 1.2.99, for example, EC 1.2.996. carboxylic acid reductase may also belong to NAD-dependent non-acylating aldehydes, such as ThnG in Sphingomonas and Cupriavidus spp ... w-transaminases may include a transaminase in EC 2.6.1, for example, EC 2.6.1.18, EC 2.6.1.19; EC 2.6.1.11; EC 2.6.1.13; EC 2.6.1.39, EC 2.6.1.48 or EC 2.6.1.62. Thioesterases can include a thioesterase in EC 3.1.2, for example, such as the YciA, tesB or AcotI3 gene product, EC 3.1.2.2; EC 3.1.2.18; EC 3.1.2.19; or EC 3.1.2.20. Thioesterases act on acyl- [acp] -thioesters include EC 3.1.2.14, such as FatA, FatB; and EC 3.1.2.21. Acid-thiol ligases or CoA synthase may include an acid-thiol ligase in EC 6.2.1, for example, EC 6.2.1.3; EC 6.2.1.5; EC 6.2.1.14; or EC 6.2.1.23. CoaA-transferases can include a CoaA-transferase in EC

2.8.3, for example, EC 2.8.3.12; EC 2.8.3.13; or EC

2.8.3.14; or the product of a CoA transferase gene in EC

2.8.3; for example EC 2.8.3.12; EC 2.8.3.13; or EC 2.8.3.14, or the ThnH gene product that catalyzes the reversible transfer of CoA to pyelic acid.

[00108] Acyl- [acp] -thioesterase includes a thioesterase in EC 3.1.2; for example, EC 3.1.2.14 and EC 3.1.2.21. Acyl- [acp] synthetase can include an acid-thiol ligase in EC

6.2.1, for example, EC 6.2.1.14, such as BioW; and EC

6.2.1.20. The amino acid aminotransferase can include an aminotransferase in EC 2.6.1, for example, EC 2.6.1.39; EC

2.6.1.21; EC 2.6.1.42; and EC 2.6.1.67. Deamination dehydration can include dehydrogenases from EC 1.4.1, for example, EC 1.4.1.2, EC 1.4.1.3; EC 1.4.1.4; and EC 1.4.1.16. Carbonyl reductases can include reductases in EC

1.1.1.184, EC 1.1.1.79; EC 1.1.1.B3; EC 1.1.1.B4 or 2-

hydroxyglutaryl dehydrogenase / alpha-ketogluterate reductases of EC 1.1.99.2 or EC 1.1.99.6.

[00109] Qa-keto acid decarboxylase can include Q & a-keto acid decarboxylases in EC 4.1.1, for example, EC4.1.1.1; 4.1.1.7; 4.1.1.72; Or an acetolactate synthase in class 2.2.1.6. The a-keto acid decarboxylase enzyme can include 4.1.1.11; 4.1.1.15; 4.1.1.16; 4.1.1.18; EC 4.1.1.20, EC 4.1.1.45; and EC 4.1.1.86.

[00110] Fatty acyl-CoA reductase can include a fatty acyl-CoA reductase in EC 1.2.1, for example, EC 1.2.1.3, EC

1.2.1.10, EC 1.2.1.22, EC 1.2.1.50; and EC 1.2.1.76. The acyl- [acp] -reductase can include the vehicle acyl-acyl-protein reductase from, for example, EC 1.2.1.80.

[00111] Aldehyde dehydrogenases can include an aldehyde dehydrogenase in EC 1.2.1, for example, EC 1.2.1.4 or EC

1.2.1.63. The aldehyde dehydrogenase / carboxylic acid reductase enzyme may also be in EC 1.2.99; e.g., EC

1.2.99.6. The carboxylic acid reductase may also belong to NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp. The carboxylic acid aldehyde dehydrogenase / reductase may also belong to the NAD-dependent non-acylating aldehyde dehydrogenase, such as ThnG in Sphingomonas and Cupriavidus spp. The aldehyde oxidase can be in EC 1.2.3, for example, EC

1.2.3.1. amidohydrolase can be an EC amidohydrolase

3.5.2, for example EC 3.5.2.11.

[00112] The hydro lyase includes a hydro lyase in EC 4.2.1, for example, EC 4.2.1.2; EC 4.2.1.59; EC 4.2.1.61; EC 4, .2.1.17 or EC 4.2.1.18. The dehydratase can also be 2-hydroxyglutaryl-CoA-dehydratase described in Clostrida and fusobacteria for which an EC number has not yet been assigned, that is, expressed with its activated (HgdCAB) or a 2-hydroxycil CoA dehydratase from an anaerobic bacterium.

[00113] Enzymes catalyzing an a-keto acid chain elongation may include one or more enzymes in the pool including AKksSA, AksD, AksE and AksF enzymes. AksA can include an AksA enzyme in EC 2.3.3, for example, EC 2.3.3.13 or EC 2.3.3.14. AksD can include an AksD enzyme in EC

4.2.1, for example, EC 4.2.1.36. AksF can include an AksF enzyme in EC 1.1.1, for example, EC 1.1.1.87. Alcohol dehydrogenases can include, for example, EC 1.1.1.1 or EC

1.1.1.2 or EC 1.1.1.21. In addition to alcohol dehydrogenases it can include, for example, Ada from Zymomonas mobilis, adhB from Zymomonas mobilis, butanol dehydrogenase from Clostridium acetobutylicum, Saccharomyces ADHIV, and ADH6 from S. cerevisiae. The enzyme alcohol oxidase can be in class EC 1.1.3.13.

[00114] Another aspect of the present invention is an isolated host cell comprising and expressing one or more exogenous nucleic acids encoding one or more exogenous nucleic acids involved in the biosynthesis of one or more polymeric monomers selected from 7 AHA; PAN; 1.7 DAH; ENTL, and 7HHA. Enzymes include ammonia lyase, enoate reductase, carboxylic acid reductases, MW-transaminases, thioesterases, acid-thiol ligases, or CoA-synthase, CoA-transferases, acyl- [acp] -thioesterases, acyl- [acp] synthetases, amino acid aminotransferase, deamination dehydrogenases, carbonyl reductases, a-keto acid decarboxylases, acetolactatosintases, Qa-amino acid decarboxylases, acyl- Coa reductases fatty, acyl- [acp] reductases, aldehyde dehydrogenase, aldehyde oxidase, hydrochloride, hydrochloride and enzymes that catalyze the elongation of a-keto acid chains. The cell and enzyme can be any of those described above for the substantially pure culture of the host cells.

[00115] Other objectives and advantages will be apparent from those technicians in the subject, from a consideration of guaranteeing the detailed descriptions represented here. Brief description of the drawings

[00116] Figure 1 is a schematic view showing the bio-transformation of pyelic acid semialdehyde, for various difunctional C7 alkanes useful for the production of nylon-7, nylon-7.7, and nylon-7, x, as well as polyesters;

[00117] Figure 2 illustrates a schematic view of the formation of semialdehyde of pyelic acid from pimeloil-CoA, pimeloil- [acp], or a-cetosuberate, all of which are intermediates in the naturally occurring routes as indicated, and the from the degradation of tetralin;

[00118] Figure 3 illustrates a schematic view of the series of Qau-keto acid chain elongation reactions. Three basic reactions are involved in chain elongation: a condensation between an a-keto diacid and acetyl-CoA to form a citrate homolog, isomerization of the citrate homolog into an isocitrate homolog, and oxidative decarboxylation of the isocitrate homolog to a Qa-keto diacid, containing an additional methylene group [Howell98: Howell Dm, Harich K, Xu H., White RH (1998). The reactions for elongation of the alpha-keto acid chain involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in methanogenic Archaea. "Biochemistry", 37 (28); 10108-17]. Successive rounds of chain elongation (3 times) from a-keto gluterate to a-keto suberate is followed by the decarboxylation of a-keto acid to pyelic acid semialdehyde. These reactions are analogous to the reactions catalyzed by citrate synthase, aconitase, and isocitrate dehydrogenase in the TCA 1 (prokaryotic) cycle and the reactions catalyzed by (R) -homocitrate synthase, hormoaconitase and dehydrogenase (-) -treo-isohomocitrate in lysine biosynthesis V [Howell, 1998, supra);

[00119] Figure 4 illustrates a schematic view of the formation of pimeloyl-CoA through the condensation of three malonyl-CoAs;

[00120] Figure 5 illustrates a schematic view of the formation of oxidative cleavage of pyelic acid from [acp] long chain fatty acid thioesters by BioI from Bacillus subtilis. Pyelic acid or its CoA or [acp] thioesters are precursors in the 7-keto-8-aminopelargonate biosynthesis in the biosynthesis route for biotin II. 7-keto-8aminopelargonate is the key intermediary for biotin biosynthesis. The degradation of pimeloyl-CoA in the host organism can be prevented by the deletion or inactivation of BioF, the gene encoding 7-keto-8-aminopelargonic acid synthase (EC 2.3.1.47);

[00121] Figure 6 is a schematic view of the formation of pimeloyl- [acp] or its methyl ester in the 7-keto-8-aminopelargonate biosynthesis in route I of biotin biosynthesis. The key intermediary in biotin biosynthesis. Pyelic acid or pyelic acid semialdehyde can be prepared from these precursors through the action of aaS (acyl-ACP synthase), FatA (acyl-ACP thiostearase A fatty) FatB (acyl-ACP thioestearase B fatty acyl-ACP thioestearase A fatty), or acyl- [acp] reductase as shown in bold text;

[00122] Figure 7 illustrates a schematic showing the formation of pimeloyl-CoA from benzoate or from the degradation of cyclohexane carboxylate;

[00123] Figure 8 is a schematic showing the formation of semialdehyde of pyelic acid in the tetralin degradation route in Sphingomonas and Corynebacterium spp;

[00124] Figure 9 illustrates a diagram showing the formation of pimeloyl-CoA in the cyclohexane carboxylate route from crotonate by methanogens. See Mouttaki et al., “Applied and Environmental Microbiology”, pages 930-938 (2001);

[00125] Figure 10A illustrates a schematic view of the examples of engineered routes for semialdehyde of pyelic acid and 7-aminoheptanoic acid from D, L-diaminopimelate as a starting point via 2-aminoheptanedioic acid. There are 2 routes from 2-aminoheptanedioic acid to pyelic acid semialdehyde, either via reductive deamination by an ammonia lyase to 2-heptanedioic acid or via the formation of Qau-keto acid which can subsequently be converted to pyelic acid or its Coa -ester. Alternatively, QO-keto pimelate derived from 2-aminoheptanedioic acid can undergo a round of chain elongation to 0-keto suberate, which can either be decarboxylated to form the pyelic acid semialdehyde, or converted to a-amino suberate, which will lead directly to 7-aminoheptanoic acid during decarboxylation. A-keto pimelate can also be derived from a-keto-adipate, which can then be derived from Ooceto-glutarate, through two successive rounds of elongation of the a-keto acid chain;

[00126] Figure 10B illustrates a schematic diagram showing the conversion of both a-hydroxypimelate and its CoA-esters to 2-heptanedioic acid or its corresponding CoA-ester, a-hydroxypimeloyl-Coa, respectively, by a hydro-lyase. Similarly, 2-heptenedioic acid or its corresponding CoaA-ester can be reduced to pyelic acid or pimeloyl-CoA, respectively, by an enoate reductase that acts on the free acid on the CoA-activated thioester. Similarly, activation of O hydroxypimelate or 2-heptenedioic acid may not be for O CoA-thioester, but unlike for [acp] -thioester by an acyl- [acp] -synthetase. Hydro-lyase can catalyze the elimination of water from a-hydroxypimeloyl- [acp], and enoate reductase can reduce the double bond of 2-heptenedioic acid- [acp]. Pimeloyl-CoA or pimeloyl- [acp] can subsequently be hydrolyzed by a transferase OR acid-thiol ligase, for recycling CoA or [acp], or by a thioesterase to produce pyelic acid;

[00127] Figure 11A shows an SDS-PAGE analysis on the soluble and insoluble fractions from the expression of the protein ammonia lyase from Citrobacter amalonaticus;

[00128] Figure 11B shows an SDS-PAGE analysis on the soluble and insoluble fractions from the expression of the protein ammonia lyase from Clostridium tetanomorphum (15) and Aspergillus oryzae (16);

[00129] Figure 12 illustrates an SDS-PAGE analysis on the soluble and insoluble fractions from the expression of the Nocardia sp carboxylic acid reductase gene;

[00130] Figure 13 illustrates the change in absorbance over time due to the reduction of pyelic acid, adipic acid, and benzoic acid to pyelic acid semialdehyde, adipic acid semialdehyde, and benzaldehyde by Nocardia's CAR reductase activity. Benzoic acid was used as a positive control and mixtures of reactions with collection of the empty vector biocatalyst as a negative control;

[00131] Figure 14 illustrates an SDS-PAGE analysis on the soluble and insoluble fractions from the expression of the protein (18) thioesterase YciA of Haemophilus influenzae;

[00132] Figure 15 illustrates an SDS-PAGE analysis of the IIvVE-Omega Vf fusion aminotransferase proteins (pING2022) and omega-optimized IIVE-Ad (pING2030);

[00133] Figure 16 illustrates the results of the bio-transformation of sodium pyruvate and 7-aminoheptanoic acid with the measured concentrations of the 7-aminohpetanoic acid substrate and the product L-alanine;

[00134] Figure 17 shows a summary of the HPLC results from the concentrations of the subterranean L- and D-2aminu acid enantiomers in the six biotransformations after 48 and 96 hours;

[00135] Figure 18 shows an SDS-PAGE analysis on soluble and insoluble fractions from the lysis of the beads of the expression of E. coli GadA proteins (129), E. coli LysA (130), E.coli GadA ilvE ( I31) and E.coli LysA ilvE (132);

[00136] Figure 19 shows the formation of T7T-aminoheptanoic acid from the decarboxylation of 2-amino suberate;

[00137] Figure 20 shows the alignment of the protein using the ClustalW algorithm showing the significant homology between MJ0277 and comD (SEQ ID 41) / come proteins (SEQ ID NO: 42);

[00138] Figure 21 shows the homology of MJ0277 to LACLA acetolactate synthase from Lactococcus lactis (SEQ ID NO: 43);

[00139] Figure 22 illustrates an analysis of the functional domains in LACLA acetolactate synthase, comD / comE from Methanocella paludicola and the hypothetical acetolactate synthase MJ0277 showing that they all have a similar structure containing a site and thiamine phosphate binding;

[00140] Figure 23 illustrates a representation of the pING2022 vector, containing the fusion gene IlvE-Omega Vf;

[00141] Figure 24 shows a representation of the vector pING2030 containing the optimized omega fusion gene I1vE-Ad;

[00142] Figure 25 represents the SEQ ID NOs sequences: 1 - 40;

[00143] Figure 26 represents a bar graph of the formation of pyelic acid from pimeloyl-Coa through the acid-thiol ligase (EC 6.2.1.5) of Thermococcus kodakaraensis (41/42), CoA transferase (EC 2.8. 3.12) of Acidominococcus fermentans (43/44) and acid-thiol ligase (EC

6.2.1.14) obtained from Pseudomonas mendocina (45); and

[00144] Figure 27 represents the formation of adiipid acid from trans-2-hexenedioic (A) and the formation of adipoyl-CoA from trans-2,3-didehydroadipoyl-Coa through the selected enoate reductases. Detailed description of the invention

[00145] In general, this description refers to methods and materials for the biosynthetic production of di- or trifunctional C7 alkanes that are useful as intermediates in the production of nylon-7, nylon-7, x, and nylon-x, 7 , as well as in production and polyesters.

[00146] In some embodiments, one or more (for example, two or more, three or more, four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; seventeen or more; seventeen or more; nineteen or more; twenty or more; or even more) isolated enzymes (for example, an ammonia lyase, enoate reductase, aminotransferase, CoA transferase, thioesterase, acid-thiol ligase, hydro-lyase, carbonyl reductase, thioesterase, carboxylic acid reductase, fatty acyl-CoA reductase, W-transaminase, a-keto acid decarboxylase , enzymes that catalyze the elongation of the a-keto acid chain, acyl-ACP thetaesterase A fatty, fatty acyl-ACP thioesterase A, FatB (fatty acyl-ACP thioesterase B), or acyl- [acp] reductase) can be used to synthetically bio-produce the di- or trifunctional C7 alkanes. Said enzymes can be isolated from recombinant cells “expressing the exogenous nucleic acids encoding the enzymes or non-recombinant cells naturally expressing the enzymes.

[00147] In some embodiments, a recombinant host that includes one or more (e.g., two or more, three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more ; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) acids exogenous nuclei encoding one or more (for example, two or more, three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more;

ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) enzymes (for example, one or more (as above) of an ammonia lyase, enoate reductase, aminotransferase, CoA transferase, thioesterterase, acid thiol ligase, hydro lyase, carbonyl reductase, thioesterase, carboxylic acid reductase , ThnG, fatty acyl-CoA reductase, O-transaminase, Q & -keto acid decarboxylase, a-amino acid decarboxylase, acetolactate synthase, amidohydrolases, enzymes that catalyze the elongation of the Qa-keto acid chain, acyl- [acp] -thioesterase, acyl- [acp] synthetase, BioW, alcohol dehydrogenase, alcohol oxidase, aldehyde dehydrogenase, aldehyde oxidase, FatA, FatB, or acyl- [acp] reductase), or a lysate prepared from said recombinant host or a cell non-recombinant naturally expressing enzyme can be used to bio-synthesize di- or tri-functional c7 alkanes. A recombinant host may have a deficiency in one or more endogenous enzymes in order to divert the metabolic intermediates towards the production of di- or trifunctional C7 alkanes.

[00148] As described herein, di-or trifunctional c7 alkanes produced bio-synthetically can be used to produce nylon-7, nylon-7, x, or nylon-x, 7, as well as polyesters. The term "nylon-7" is a polyamide produced by the polymerization of the 7-aminoheptanoic acid monomer or by opening condensation of the enantolactam ring. [o] nylon-7 is also known as "polyienantamide" or "polyheptanamide".

[00149] The term "nylon-x, 7" refers to the family of polyamides that are produced by the polymerization of heptane-1,7 dioic acid (also known as pyelic acid or pimelate) with a diamine. The integer x indicates the number of carbon atoms in the diamine with which heptane-1,7-dioic acid is reacted. In some embodiments, the integer x is an integer greater than 3, for example, greater than 5, greater than 7, or greater than 9. For example, nylon-5,7 is produced by the reaction of heptane-1,7. -diocese and 1,5- pentanediamine. Nylon-7,7 is produced by the reaction of heptane-1l, 7-dioic acid and 1,7-diaminoheptane.

[00150] The term “nylon-7, X”, refers to the family of polyamides that are produced by the polymerization reaction of heptanethylene diamine with a dicarboxylic acid. The integer x indicates the number of carbon atoms in the dicarboxylic acid with which heptanethylenediamine is reacted. For example, nylon-7,5 is produced by the reaction of heptamethylenediamine (also known as 1.7 diaminoheptane or 1,7-heptanediamine) and pentane-1, 5-dioic acid. In some embodiments, the integer x is an integer greater than 3, for example, greater than 5, greater than 7 or greater than 9.

[00151] The term "polyester" refers to a category of polymers that contains an ester of the functional group in its main chain. Polyesters can be homopolymers, for example, polycaprolactone, that is, produced by ring opening condensation. Polyester copolymers are formed through condensation by esterification of polyfunctional alcohols and acids. For example, adipic acid and ethylene glycol are used to produce polyethylene adipate. Pyelic acid can be used in place of adipic acid in said copolymers. Alternatively, 7-hydroxyheptanoic acid can be used to produce polyanantolactone.

[00152] The above and other aspects of the present invention will now be described in more detail with respect to the embodiments described here. It should be appreciated that the invention can be configured in different forms and should not be construed as limited to the embodiments depicted here. In particular, these embodiments are provided so that this description will be finalized and completed, and will lead to the scope of the invention for those skilled in the art.

1.1. Definitions:

[00153] The terminology used in the description of the invention is here for the purpose of particular description of the descriptions only and is not intended to limit the invention. As used in describing the embodiments of the invention and in the appended claims, the singular form "one", "one" and "o" "a" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, as used herein, “and / or” refers to, and covers any and all possible combinations of one or more of the associated items listed. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[00154] As used here, the term "enzyme" or "enzymes" refers to a protein catalyst capable of catalyzing a reaction. Here, the terms not only mean an isolated enzyme, but also include a host cell expressing that enzyme. Consequently, the conversion of A to B via enzyme C must also be constructed to encompass the conversion of A to B via a host cell expressing enzyme C.

[00155] As used herein, the term "difunctional C7 alkane" refers to a linear or branched, saturated or unsaturated hydrocarbon, having seven carbon atoms and two functional groups. Functional groups can be positioned anywhere near the hydrocarbon chain. In some embodiments, the functional groups are located at the terminal ends of the hydrocarbon chain (for example, a functional group at each terminal). The term is also intended to encompass cyclic compounds such as enantolactam. Examples of difunctional but not limited to C7 alkanes, Ppimeloil-CoA, pimeloyl- [acp], heptane-1,7-dioic acid (pyelic acid or pimelate), 7-aminohpetanoic acid, 7T-hydroxyheptanoic acid, heptamethylene diamine, 1,7-heptanediol, 7-aminoheptanol, 7-aminoheptanal, enantolactam, pyelic acid semialdehyde (also known as 7-oxoheptanoate), 2-heptenediacid, and 2-diacid-CoA.

[00156] As used here, the terms "-oate" and "oic" acid, for example, heptanoate and heptanoic acid, are used interchangeably. In addition, it should be understood that "-ate", "-oate" and "-oic" acids can be used interchangeably through the application to refer to compounds in any of their neutral or ionized forms, and the so-called forms “Zwiterionics”.

[00157] As used herein, the term "trifunctional c7 alkane" refers to linear or branched, saturated or unsaturated hydrocarbon, having seven carbon atoms and three functional groups. Functional groups can be positioned anywhere along the hydrocarbon chain. In some embodiments, two of the three functional groups are located at the terminal ends of the hydrocarbon (for example, a functional group at each terminal). Examples of tri-functional C7 alkanes include, but are not limited to, 2-oxopimelate (0-ketopimelate), 2-aminopimelate (a-aminopimelate), 2-hydroxypimelate (a-hydroxypimelate), and 2-hydroxypimelate-CoA (a-hydroxypimelate -CoA).

[00158] As used herein, the term "functional group" refers to an amine group, a carboxylic acid group, an aldehyde group, an alcohol group, an A co-enzyme group or a keto group. The term 'amino group' refers to a radical - NH2. The term "carboxylic acid group" refers to a -COOH radical. The term "aldehyde group" refers to the radical -C (O) H. the term "alcohol group" refers to the radical -OH. The term "keto group" refers to the radical C (O).

[00159] The term "coenzyme group A" refers to the organic cofactor, or prosthetic group (non-protein portion of an enzyme), whose presence is required for the activity of many enzymes (apoenzyme) to form an active enzyme system. The functions of coenzyme A in certain condensation enzymes, act on the transfer of the acetyl group or another acyl group and on the synthesis of fatty acid and oxidation, oxidation and pyruvate and in other acetylation.

[00160] The term "acp" or "vehicle acyl protein" refers to a protein (which is typically active in the synthesis of fatty acid) that joins the acetyl and malonyl groups from coenzyme A to acetyl and coenzyme A to molonyl and connects them by condensation to form the B-keto acid acyl carrier protein, releasing carbon dioxide and the sulfhydryl form of the acyl carrier protein.

[00161] The functional groups in a di- or trifunctional C7 alkane can be the same or a different one. For example, in some embodiments, with a difunctional C7 alkane, both functional groups can be amino groups, both functional groups can be carboxylic acid groups, or both functional groups can be alcohol groups.

[00162]

[00163] In some embodiments, one of the functional groups in a difunctional C7 alkane is an amino group and the other is a carboxylic acid group. In some embodiments, one of the functional groups in a difunctional C7 alkane is an alcohol group and the other is a carboxylic acid group. In some embodiments, one of the functional groups in a difunctional C7 alkane is an alcohol group and the other is an amino group.

[00164] In some embodiments, with a trifunctional c7 alkane, two of the functional groups are carboxylic acid groups and a functional group is a keto group or a hydroxyl group or an amino group.

[00165] The term "heterologous" is used here to refer to any nucleic acid or polypeptide that is not derived from a cell of the same species as that of the host cell. Consequently, as used here, "homologous" nucleic acids or proteins are those that occur in, or are produced by, a cell of the same species as the host cell.

[00166] The term "exogenous" as used herein with reference to nucleic acid and a particular host cell refers to any nucleic acid that does not occur in (and cannot be obtained from) that particular cell as found in nature.

Thus, a non-naturally occurring nucleic acid is considered to be exogenous to a host cell once it is introduced into the host cell.

It is important to note that non-naturally occurring nucleic acids can contain substrates of nucleic acid or fragments of nucleic acid sequences that are observed in nature providing that the nucleic acid as a whole does not exist in nature.

For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is a non-naturally occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host cell, since the molecule nucleic acid as a whole (genomic DNA plus DNA vector) not found in nature.

Thus, any vector, autonomously replicating plasmid, or virus (for example, retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be a non-naturally occurring nucleic acid.

In the sequence, that fragment of genomic DNA produced by PCR or restriction endonuclease treatment as well as cDNAsS are considered to be non-naturally occurring nucleic acids containing a promoter sequence and the polypeptide coding sequence (for example, cDNA or genomic DNA) in an arrangement not found in nature is an unnaturally occurring nucleic acid.

A naturally occurring nucleic acid can be exogenous in a particular cell.

For example, an entire chromosome isolated from a yeast cell x is an exogenous nucleic acid relative to a yeast cell since the chromosome is introduced into a yeast cell.

[00167] It will be clear from the above that "exogenous" nucleic acids can be "homologous" or "heterologous" nucleic acids. In contrast, the term “endogenous” as used here with reference to nucleic acids or genes (or proteins encoded by nucleic acids or genes) and a particular cell refers to any nucleic acid or gene that does not occur in (and can be obtained from, from that) particular cell as found in nature.

[00168] As used here, the term "recombinant host" refers to a host, the genome of which has been grown on at least one exogenous nucleic acid sequence. Said nucleic acid sequences include, but are not limited to, nucleic acids that are naturally present in the host (i.e., a heterologous nucleic acid), DNA sequences that are not normally transcribed within RNA or translated into a protein (“ expressed ”), and other nucleic acid sequences whose desire is to introduce into the non-recombinant host (for example, a regulatory region to alter the expression of a particular nucleic acid sequence). It should be appreciated that the genome of a recombinant host described here is typically grown by the stable introduction of one or more exogenous nucleic acids. Generally, exogenous nucleic acid is not originally resident in the host that is the DNA receptor (i.e., it is a heterologous nucleic acid), but is within the scope of the invention to isolate a nucleic acid from a given host, and to subsequently introduce one or more additional copies of that nucleic acid into the same host, for example, to improve the production of an encoded product or to change the expression pattern of a nucleic acid. In some instances, the nucleic acid will modify or even replace an exogenous gene or DNA sequence with, for example, homologous recombination or site-directed mutagenesis. The modification or replacement of an endogenous gene can result in a deficiency in a particular encoded product, for example, an enzyme. Suitable recombinant hosts are described below. Recombinant hosts may also contain nucleic acids that are not incorporated into the host cell's genome, but exist (and preferably replicate) episomally in the cell.

[00169] As used here, a "promoter" refers to a DNA sequence that enables a gene to be transcribed. The promoter is recognized by an RNA polymerase, which then initiates transcription. Thus, a promoter contains a DNA sequence that is either directly linked by, or is involved in, recruitment of the RNA polymerase. A promoter sequence can also include "enhancer regions" that are one or more regions of DNA that can be linked with proteins (nominally, the transcription factors, such as a set of transcription factors) to improve the level of transcription of the genes (hence the name) in a gene group. An enhancer, although typically at the 5 'end of a coding region, can also be separated from a promoter sequence and can be, for example, within an intronic region of a gene or 3' / for the coding region of the gene.

[00170] As used here, "operably linked" means incorporated within a genetic construct so that expression control sequences effectively control the expression of a coding sequence of interest.

1.2 Raw material:

[00171] A variety of different raw materials can be used to produce di- or tri-functional alkanes. In some embodiments, a recombinant host cell, or a lysate prepared from a cell (for example, a recombinant host cell), is contacted with a renewable raw material to produce a di- or trifunctional C7 alkane, such as acid 7 -aminoheptanoic. Examples of renewable raw material that can be used as a carbon source include, but are not limited to, cellulosic raw material, plant-derived carbohydrates and fatty acids. In some embodiments, the raw material with which the recombinant host cell is in contact is glucose, sucrose, xylose, fatty acids or glycerol.

[00172] In addition to the renewable raw material, such as a synthesis gas ("syngas"), such as those listed above, as a carbon source. In this specific embodiment, the recombinant host cells of the invention are constructed to provide an efficient metabolic route for using synthesis gas, or another source of carbon gas, to produce di- or trifunctional C7 alkanes.

[00173] Additionally, the recombinant hosts described here, or a lysate prepared from the recombinant host cell, can be contacted with a raw material

aromatic hydrocarbon material. The use of aromatic hydrocarbon raw material, in addition to the renewable raw material, provides a way to convert the aforementioned petroleum-based material into compounds that are more benign to the environment, reducing impact to the environment. Examples of suitable aromatic hydrocarbon feedstock include, but are not limited to, toluene, benzene, benzoic acid and chiquimate (“shikimate”).

1.3 Routes to the difunctional C7 alkanes comprising enzyme catalysis steps:

[00174] As stated above, embodiments of the present invention pertain to methods for producing di- or trifunctional C7 alkanes in the presence of one or more enzymes isolated in the presence of a recombinant host cell expressing that / those enzyme (s), or in the presence of a cell lysate (or a partially purified lysate) of a cell (e.g., recombinant cell) that expresses the enzyme (s). In some embodiments, the production of di- or trifunctional C7 alkanes starts with pimelate or pimeloil-Coa Or pimeloil- [acph] and proceeds to a common intermediate, called pyelic acid semialdehyde. Specifically, in some embodiments, the invention relates to a method for producing difunctional C7 alkane from 7-aminoheptanoic acid through the use of an enzyme that catalyzes the amination and semialdehyde of pyelic acid to 7-aminoheptanoic acid. See, figure 1. A semialdehyde aminotransferase, such as a QO-transaminase, can be used. Examples of o-transaminase include, but are not limited to, transaminases classified under EC 2.6.1 such as EC

2.6.1.18; EC 2.6.1.19; EC 2.6.1.1; EC 2.6.1.13; EC 2.6.1.48;

and EC 2.6.1.62. In some embodiments, the aminotransferase is B-alanine aminotransferase classified in EC 2.6.1.18 from v. fluvialis, B. weihenstephanensis, P. aureginosa, B. subtilis, or P. syringae. See WO 2011/031147, which is incorporated here in its entirety.

[00175] In some embodiments, 7-aminoheptanal can be produced using an enzyme that catalyzes the dehydrogenation of 7-aminoheptanoic acid aldehyde to 7-aminoheptanal. See, figure 1. An aldehyde dehydrogenase classified under EC

1.2.1, such as EC 1.2.1.4 or EC 1.2.1.63, can be used to produce 7T-aminoheptanal from 7T-aminoheptanoic acid. Carboxylic acid aldehyde dehydrogenase / reductase may also be in EC 1.2.99, for example, EC

1.2.99.6. Carboxylic acid reductase may also belong to NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus sSpp ..

[00176] In some embodiments, heptamethylenediamine (also known as 1,7-diaminoheptane) can be produced from 7-aminoheptanal using an enzyme that catalyzes the transfer of an amino group. See Figure 1. Suitable aminotranserases include, but are not limited to, aminotransferase in EC 2.6.1 such as EC 2.6.1.18; EC

2.6.1.19; EC 2.1.11; EC 2.6.1.13; EC 2.6.1.39; EC 2.6.1.48; and EC 2.6.1.62. In some embodiments, the aminotransferase is B-alanine aminotransferase classified in EC 2.6.1.18 from V. fluvialis, B. weihenstephanensis, P. aureginosa, B. subtilis, or P. syringae. See publication WO 2011/031147, which is incorporated here in its entirety.

[00177] In some embodiments, enantolactam can be produced from 7-aminoheptanoic acid using an enzyme that catalyzes the amide hydrolysis of 7-aminoheptanoic acid. See, figure 1. Suitable amidohydrolases include amidotransferases classified under EC

3.5.2 such as EC 3.5.2.12 or EC 3.5.2.11 can be used.

[00178] In some embodiments, 7T-hydroxyheptanoate is produced from pyelic acid semialdehyde or 7-aminoheptanol is produced from 7-aminoheptanal using an enzyme that catalyzes the reduction of an aldehyde. See the figure

1. For example, an alcohol dehydrogenase classified under EC

1.1.1. such as EC 1.1.1.2 or EC 1.1.1.21 can be used. It should be understood by technicians on the subject that there is a greater amount of alcohol dehydrogenase with broad ranges of substrates, and that other dehydrogenases from EC

1.1.1.-; such as 1.1.1.B3 or EC 1.1.1.B4, or 1.1.1.79, or in EC 1.1.99, such as EC 1.1.99.2 or EC 1.1.99.6, can also be used to reduce an aldehyde in the corresponding alcohol . Alternatively, an alcohol oxidase can also be used. Alcohol oxidase can be in class EC 1.1.3.13.

[00179] In some embodiments, 1,7-heptanediol can be produced from 7-hydroxyheptanoate using an aldehyde dehydrogenase and an alcohol dehydrogenase. An appropriate aldehyde dehydrogenase can be classified under EC 1.2.1 (for example, 1.2.1.4 or EC 1.2.1.63 or EC 1.1.1.21). See figure 1. An alcohol dehydrogenase can be classified under EC 1.1.1. such as EC 1.1.1.1 or EC 1.1.1.2. It should be understood by those skilled in the art that a greater number of alcohol dehydrogenase with broad ranges of substrates, and that other dehydrogenases of EC 1.1.1.-, such as 1.1.1.B3 or EC

1.1.1.B4, or EC 1.1.1.79, or in EC 1.1.99, such as EC

1.1.99.2 or EC 1.1.99.6, can also be used to reduce an aldehyde in the corresponding alcohol. Alternatively, an alcohol oxidase may be in the Ec class

1.1.3.13. Similarly, a vast number of aldehyde dehydrogenases or carboxylic acid reductases with broad substrate specificities exist. Suitable aldehyde dehydrogenases include EC 1.2.1.3; EC 1.2.1.4; and EC 1.2.1.63. The aldehyde dehydrogenase / carboxylic acid reductases can also be from EC 1.2.99, for example, EC

1.2.99.6. The carboxylic acid aldehyde dehydrogenase / reductase may also belong to NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp .. Alternatively, an aldehyde oxidase can be used, such as an aldehyde oxidase obtained from EC

1.2.3.1.

[00180] In embodiments in which di- or trifunctional C7 alkanes can be toxic to the host (e.g., semialdehyde such as 7-oxoheptanoate or 7-aminoheptanal), conversion to the toxic compound can be performed in vitro using isolated enzymes or a lysate from the recombinant host. In some embodiments, the toxic compound is produced by the host and then subsequently converted (using isolated enzymes, a recombinant host expressing an enzyme, or chemically converted) to enantolactam.

1.5 Biosynthetic route for pimelate and / or pimeloil-CoA:

[00181] Pimelate and / or pimeloyl-CoA can be obtained via a number of different pathways including: (i) suberate a-keto produced from the a-keto acid chain elongation route, or (ii) the biotin biosynthesis; (iii) biotin II biosynthesis route; (iv) condensation and three molecules of malonyl-CoA in a single molecule of pimeloyl-CoA; (v) the benzoate degradation route; (vi) cyclohexane-carboxylate route, (vi) the D, L-diaminopimelate route; and (vii) a biosynthetic route from the carbon source is crotonate. See figure 2. These routes for pimelate and / or pimeloil-CoA or pimeloil- [acph] are described in more detail below.

[00182] In some of these routes, pimelate can be produced from pimeloyl-CoA using an enzyme that catalyzes the hydrolysis of a thioester. For example, enzymes that can hydrolyze a thioester include, but are not limited to, thioesterases, acid-thiol ligases, and CoA transferases. Suitable thioesterases include thioesterases classified in EC 3.1.2 such as the YciA, tesB or Acotl3 gene product; EC 3.1.2.2; EC 3.1.2.18; EC 3.1.2.19 or EC 3.1.2.20. Thioesterases acting on acyl- [acp] thioesters include EC

3.1.2.14, such as FatA, FatB, and EC 3.1.2.21. For example, one of the following enzymes can be used. A thioester-hydrolase / acyl-CoA thioesterase classified in EC 3.1.2 such as an ADP-dependent middle chain acyl-CoA hydrolase in EC 3.1.2.19 and EC 3.1.2.18, an acyl-CoA hydrolase in EC

3.1.2.20; or the YciA, tesB or Acotl3 gene product; oleoyl- [acyl-protein vehicle] hydrolases in EC 3.1.4.14. Suitable acid-thiol ligases include thiol acid ligases classified in EC 6.2.1 such as EC 6.2.1.3; EC 6.2.1.14; and Ec 6.2.1.23. Suitable CoaA transferases include CoA transferases classified under EC 2.8.3 such as EC 2.8.3.6; EC 2.8.3.8; EC 2.8.3.12; EC 2.8.3.13 or EC 2.8.3.14, or the ThnH gene product that catalyzes the reversible transfer of CoA in pyelic acid.

[00183] Heptanol, 7-dioic acid can be obtained from pimeloyl- [acp] through the use of enzymes acting on [acp] -thioesters, such as acyl [acp] | -thioesterases in EC

3.1.2.14, such as fatA and fatB or EC 3.1.2.21. Alternatively, acid-thiol ligases acting on [acp] - thioesters can be used, such as, EC 6.2.1.14 and Ec

6.2.1.20.

[00184] Alternatively, pyelic acid can be obtained from the pimeloyl- [acp] 1-methyl ester by hydrolysis of the ester bond by AsaS (acyl-ACP-synthetase), followed by removal of the methyl group by a lipase / esterase.

[00185] Pimelate can be transformed into semialdehyde of pyelic acid using an enzyme that catalyzes the reduction of a carboxylic acid. Appropriate reductases include, but are not limited to, carboxylic acid reductases classified in EC 1.2.99. Carboxylic acid reductases may also belong to NAD-dependent non-acylating aldehyde dehydrogenases, such as ThnG in Sphingomonas and Cupriavidus spp .. or fatty acyl-CoA reductases classified in EC 1.2.1., Such as EC 1.2.1.3; Ec 1.2.1.10; EC 1.2.1.22; EC 1.2.150; EC 1.2.1.57; and EC 1.2.176. In some embodiments, a fatty acyl-CoA reductase can directly convert pimeloyl-CoA or pimeloyl- [acp] to pyelic acid semialdehyde. Examples of carbonyl reductase in EC 1.2.99 include, but are not limited to, EC

1.2.99.6 from Norcardia sp. (Aimin et al., “Appl. Environ. Microbiol.” 70, 1874-1881 (2004), incorporated by reference) or S. griséus (Suzuki et al., “J. Antibiot.”, (Tokyo), 60 : 380-387 (2007) incorporated by reference).

Examples of fatty acyl-CoA reductases in EC 1.2.1 include, but are not limited to, EC 1.2.1.75, from, for example, C. aurantiacus (Hugler, M., et al., “J.Bacteriology”, 184: 2404-2410 (2002), incorporated herein by reference), M. sedula (Kockelkorn, D. and Fuchs, G., “JJ. Bacteriology”, 191: 6352-6362 (2009), incorporated by reference), or Ss. tokodai (Alber, B. et al., "OT. Bacteriology", 188: 8551-8559 (2006), incorporated herein by reference); and 1.2.1.50 obtained from, for example, Acinetobacter sp., A. platyrhynchos (Ishige, T., et al., “Appl. Envtl. Microbiology”, 68: 1192-1195 92002), incorporated herein by reference), A thaliana (Doan, TT, et al., “Journal Plant Physiology”, 166: 787-796 (2009) and Hooks, MA, et al. “Plant J.”, 20: 1-13 (1999), both which are incorporated by reference); Homo sapiens (McAndrew, RP, et al., “J. Biol. Chem. 283: 9435-9443 (2008), incorporated by reference), M. Musculis, P. leiognathi, P. phosphoreum, P. sativum, or S Chinese. Examples of acyl- [acp] reductases in EC 1.2.1 include EC 1.2.1.80, from, for example, Synecoccus elongates (Shirmer, A. et al. (2010). “Microbila Biosynthesis of alkanes - Science”, 329 (5991: 559-562).

1.5.1 Elongation of the a-keto acid chain:

[00186] Difunctional alkanes can be produced from Qa-keto glutarate via successive chain-length reactions to adipate 0-keto, pimelate aceto or suberate a-keto known in the art. See, for example, figure 3 and publication WO 2010/068944. The decarboxylation of α-keto dioic acid (Cn, n = 5-8) or the corresponding 2-amino-dioic acid then provides precursors for Cn-1 difunctional O, OW-alkanes, for example, C4-7. These successive alpha-keto acid chain elongation reactions occur on the coenzyme B biosynthesis pathway as elucidated in the methanogenic bacteria, such as -. Methanocaldococcus jannaschii, Methanococcus voltae, and Methanosarcina thermophila.

[00187] These three successive rounds of chain stretching from Qa-keto glutarate (C5) to a-keto suberate are catalyzed through the same three enzymes acting three times (and in the case of EC 4.2.1.114 (AksD (large subunits of 3-isopropylmalate dehydratase) / AksE (small subunits of 3-isopropylmalate dehydratase) six times) on the substrates, each time increasing the chain length, see figure 3. These enzymes include, but are not limited to: EC 2.3.3.14 : homocitrate synthase / AksA (alpha-isopropylmalate synthase), which acts on a-ketoglutarate, a-keto adipate, and a-keto imelate; EC 4.2.1.114; homoaconitase or AksD / AKksSE, which catalyzes dehydration reactions of ( R) -homocitrate, dihomocitrate and trihomocitrate, as well as hydration reactions of cis-homoaconitate, cis- (homo); - aconitate and cis- —- (homo); -aconitate; and Ec 1.1.1.87: dehydrogenase homoisocitrate or AksF (multifunctional 3-isopropylmalate dehydrogenase / D-malate dehydrogenase; which catalyze the oxidative xylation dependent on NAD (P) + homoisocitrate, treo-tha (homo),; - citrate and treo-tha (homo); - citrate.

[00188] Examples of AksA enzymes include, but are not limited to, those in EC 2.3.3 such as EC 2.3.13 or

2.3.3.14. Examples of AksD enzymes include those in EC

4.2.1 such as EC 4.2.1.36. Examples of AksF enzymes include, but are not limited to, the enzymes in EC 1.1.1., Such as, EC

1.1.1.87. Crotonil-CoA is biotransformed into glutaconyl-1-Coa using an enzyme classified under EC 4.1.1.70; glutaconyl-l1- Coa is biotransformed into glutaryl-CoA using an enzyme classified under EC 1.3.99.7; glutaryl-CoA is biotransformed to 3-ketopimelyl-CoA using an enzyme classified under EC

2.3.1.43; 3-ketopimelyl-Coa is biotransformed to 3-hydroxypimelyl-Coa using an enzyme classified under EC

1.1.1.4259; 3-hydroxypimelyl-CoA is biotransformed to 2,3-didehydro-pimeloyl-CoA is biotransformed to pimeloyl-CoA using an enzyme classified under EC 1.3.1.62. Pimeloyl-Coa is subsequently transformed into pyelic acid.

[00189] Thus, in some embodiments, the invention provides a recombinant host cell comprising enzymes for converting acetyl-CoA to heptane-1,7-dioic acid. In some embodiments, the recombinant host cell has a deficiency in 7-keto-8-aminopelargonic acid synthetase, an enzyme capable of converting 6-carboxyhexanoyl-CoA (pimeloyl-CoA) into 7-keto-8-aminopelargonic acid (KAPA), and encoded by the BioF gene in the host, if the host organism has a native biotin biosynthesis pathway, so that pimeloil-Coa or Ppimeloil- [acp] cannot be transferred in biotin biosynthesis. In some embodiments, BioI is overexpressed to increase the pimeloyl- [acp] formed via oxidative cleavage of fatty acid- [acp] esters. In some embodiments, [lacp] - transferase / synthetases activates the long fatty acid chains for their corresponding [acp] -thioesteres for cleavage by overexpressed BioI in the host cell. Embodiments of the invention also provide a method for producing heptane-1,7,7-dioic acid comprising the use of said recombinant host cells.

1.5.6 benzoyl-CoA degradation route:

[00190] An additional source of pimeloil-Coa and / or pyelic acid contemplated here is the benzoate degradation route in figure 7. See, for example, Bernsteinb, JR, et al., “Metabolic Eng'9”, 10: 131-140 (2008); Harwood, C.S., et a., "FEMS, Microbiology Reviews", 22: 439-458 (1999); and Harrison, F.H., and Harwood, C.S., "Microbiology", 151: 727-736 (2005), all of which are incorporated herein by reference. A number of transformations are illustrated in figure 7, where the benzoate is converted to benzoyl-CoA using an enzyme classified under EC 6.2.1.25, and ending in pimeloyl-Coa. Benzoyl-CoA is converted to cyclohexa-1,5-dienocarbonyl-CoA using an enzyme classified under EC 1.3.99.15. In some embodiments, cyclohexa-1,5-dienocarbonyl-CoA is biotransformed to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA using an enzyme classified under EC 4.2.1.100; o-hydroxycyclhex-1-ene-1-carboxyl-CoA is biotransformed to 6-cetoxicyclohex-1-ene-1-carboxyl-CoA using an enzyme classified under EC 1.1.1-; 6-cetoxicyclohex-1-ene-carboxyl- Coa is biotransformed to 3-hydroxypimelyl-Coa using an enzyme classified under EC 3.7.1.-; 3-hydroxyimelyl-CoA is biotransformed to 6-carboxylhex-2enoyl-CoA using an enzyme classified under EC 4., 2.1-; 3-hydroxypimelyl-CoA is biotransformed into pomeliol-CoA using an enzyme classified in EC 1.3.1.62. Pimeloyl-CoA, on the way back, is converted to pyelic acid (heptane-1,7,7-dioic acid). In some embodiments, cyclohex-1,5-dienocarbonyl-CoA is biotransformed into cyclohex-1-ene-lcarboxyl-CoA, cyclohex-1-ene-1-carboxyl-CoA is biotransformed into 2-hydroxycyclohexane-

l1-carboxyl-CoA using an enzyme classified under EC 4.2.1.-; 2-hydroxycyclohexane-1-caroxyl-Coa is biotransformed to 2-ketocyclohexane-1-carboxyl-CoA is biotransformed into pimeloyl-CoA using an enzyme classified in EC 3.1.2.- (for example, a -ketocyclohexane-carboxyl-CoA hydrolase Rp-badI, an enzyme classified under EC 3.1.2.-).

[00191] In some embodiments, the recombinant host cell capable of producing heptane-1,7-dioic acid includes one or more of the enzymes listed in figure 7 or in this section.

1.5.7 2,6-diaminopimelate route:

[00192] Yet another source of pimelate is D, L-diaminopimelate, also known as 2,6-diaminopimelate. D, L-diaminopimelate is an intermediate in the production of lysine. In some embodiments, an endogenous lysine pathway can be modified to produce, preferably, pimelate by breeding and recombinant host cells that have a deficiency in diaminopimelate decarboxylase, see, for example, figure 10. AD, L diaminopimelate can be biotransformed into a monoamino-pimelic acid unsaturated by an enzyme that catalyzes the reductive deamination of D, L-diaminopimelate in 6-amino-2-heptenedioic acid. Examples of enzymes that catalyze the reductive deamination of D, L-diamino-pyylate include ammonia lyases classified in EC 4.3.1 such as EC 4.3.1.1; EC 4.3.1.2; EC 4.3.1.3; EC 4.3.1.9; EC 4, .3.1.12; EC 4.3.1.13; EC 4.3.1.14; EC 4.3.1.23; and EC

4., 3.1.24. In some embodiments, ammonia lyase is an ammonia lyase methyl aspartate classified under EC 4.3.1.2 from C. amalonaticus, C. tetanomorphum or A. oryzae. See, for example, Botting, “Biochemistry”, 27: 2953-2955

(1988); and Kato, “Appl. Microbiol. Biotechnol. ”, 50: 468-474 (1998), the total of which is incorporated herein by reference.

[00193] 6-Amino-2-heptenedioic acid can be biotransformed into 2-amino-2-heptenedioic acid using an enzyme that catalyzes the enoate reduction of 6-amino-2-heptenedioic acid to 2-amino-2-heptanoedioic acid ( also known as 2-aminopimelic acid or Qaraminopimelate). Examples of enzymes that catalyze the reduction of 6-amino-2-heptenedioic acid enoate include enoate reductases classified in EC 1.3.1, such as EC 1.3.1.8; EC 1.3.1.9 EC 1.3.1.10; EC 1.3.1.31; EC 1.3.1.38; EC 1.3.1.39; EC

1.3.1.44 or pimeloyl-CoA dehydrogenase in EC 1.3.1.62, such as PimC / PimD or ThnJ / ThnK. The enoato-reductase can also be in Ec 1.3, such as 1.3.8.1 or Ec 1.3.99.3; EC 1.3.99.B10 or Syntrophys aciditrophicus 2,3-didehydropyelyl-CoA reductase and homologues thereof. In some embodiments, the enoate reductase is YqiM or OPR1 / 3 in EC 1.3.1.31 from B. subtilis or L. esculentum, respectively. See, for example, Stueckler, “Org. Lett. ”, 9: 5409-5411 (2007) Ktzing,“ J. Biol. Chem. ”, 280: 27904-27913 (2005); Hall, “Angew. Chem. Int. Ed. ”, 46: 3934-3937 (2007); and Breithaupt, “Proc. Natl. Acad. Sci. ”, USA 103: 14337-14342 (2006), which is incorporated here in its entirety.

[00194] 2-Aminopimelic acid can be converted into the corresponding 2-heptenedioic acid (also known as 2,3-didehydropyelic acid) through an enzyme that catalyzes the reductive deamination of 2-aminopimelic acid. Examples of enzymes that catalyze the reductive deamination of 2-aminopimelic acid include ammonia lyases classified as

EC 4.3.1 such as EC 4.3.1.1; EC 4.3.1.2; EC 4.3.1.3; EC

4.3.1.9; EC 4.3.1.12; EC 4.3.1.13; EC 4.3.1.14; EC 4.3.1.23; or EC 4.3.1.24, as described above.

[00195] 2-Heptene dioic acid can be transformed into 2-heptene-dioic acid-CoA using a CoA transferase (for example, a CoA transferase classified in EC 2.8.3, such as EC 2.8.3.12; EC 2.8 .3.13 or EC 2.8.3.14 or the ThnH gene product that catalyzes the reversible transfer of Coa to pyelic acid) and an acid-thiol ligase (for example, an acid-itol ligase classified in EC 6.2.1, such as EC

6.2.1.3; EC 6.2.1.5; EC 6.2.1.14; or EC 6.2.1.23).

[00196] 2-Heptene dioic acid-CoA is then converted to pimeloyl-CoA by an enzyme that catalyzes the enoate reduction of 2-heptene dioic acid such as an enoate reductase enzyme classified in EC 1.3.1. Pimeloyl-CoA can be converted to pimelate or semialdehyde of pyelic acid as described above.

[00197] As shown in figure 10 and figure 3, QO-keto pimelate can also pass a chain-lengthening run on a-keto-suberate using chain-lengthening enzymes, such as those present in methanogenic bacteria (for example , methanobacterium autotrophicum). Said chain elongation enzymes, but are not limited to AksA, AksD, and AksSE or F as discussed above. Examples of AksA enzymes include, but are not limited to, those in EC 2.3.3. Examples of enzymes in EC 2.3.3 include, but are not limited to, EC 2.3.13 or 2.3.3.14. Examples of AksD enzymes include those in EC 4.2.1 An exemplary enzyme in EC 4.2.1 includes, but is not limited to, EC 4.2.1.36. Examples of AksF enzymes include, but are not limited to, in EC 1.1.1. An example of an enzyme in EC 1.1.1. It includes, but is not limited to, EC 1.1.1.87. And in this example, the enzyme representative comprises a decarboxylase. Examples of decarboxylase include those in EC 4.1.1. Examples of decarboxylases in EC 4.1.1 include, but are not limited to, EC 4.1.1.11; EC 4.1.1.15; EC 4.1.1.17; EC 4,1.1.18; EC 4.1.1.19; EC 4.1.1.20 and EC 4.1.1.86.

[00198] In some embodiments, acetyl-CoA can be fed in a cycle of tricarboxylic acid (TCA), in which acetyl-CoA is transformed into succinic acid (butan-1,4-dioic acid). Succinic acid is then funneled from the TCA cycle into a synthesis of pyelic acid starting with the bio-transformation of 2-oxoglutarate into 2-hydroxy-1,2,4-butanotricarboxylic acid (homocitrate) (for example, an enzyme in EC 2.3.3.14); bio-transformation of homocitrate to homo-cis-aconitase into homo-cis-aconitase to homoisocitrate using an enzyme classified under EC 4.2.1.36; bio-transformation from homoisocitrate to oxaloglutarate using an enzyme classified under EC 1.1.1.87; bio-transformation of oxaloglutarate to 2-oxoadipate using an enzyme classified under EC 1.1.1.87, and bio-transformation of 2-oxodipate to glutaryl-CoA using an enzyme classified under EC 1.2.4.2. Glutaryl-CoA can be converted to pomeloyl-CoA as discussed above. The homocitrate is finally converted to pimeloyl-CoA and subsequently to pyelic acid as described here.

[00199] α-keto-suberate can also be decarboxylated to an α-keto decarboxylase acid to form pyelic acid semialdehyde, or converted to α-aminosuberate, using an amino aminotransferase that will lead directly to T7-aminoheptanoic acid in decarboxylation by an α-amino decarboxylase acid. Alternatively, pyelic acid semialdehyde can be converted to 7-aminoheptanoic acid using a Q-aminotransferase. Examples of Qa-keto acid decarboxylases include those in EC 4.1.1. such as EC

4.1.1.1; EC 4.1.1.7; and EC 4.1.1.72 or an acetolactate synthase in class EC 2.2.1.6.

[00200] Examples of a-amino acid decarboxylase include those in EC 4.1.1.11; EC 4.1.1.15; EC 4.1.1.17; EC 4, .1.1.18; EC 4.1.1.19; EC 4.1.1.20; EC 4.1.1.45 and EC

4.1.1.86. In some embodiments, the decarboxylases comprise kivD keto-isovalerate decarboxylase in EC 4.1.1.1 of L. lactis; benzoylformate decarboxylase mdIC A460I or BFD in EC 4.1.1.7 of P. putida; isozymes decarboxylase pyruvate 1 and 2 pdel in EC 4.1.1.1 of S. cerevisiae; pdce pyruvate decarboxylase (1472A) from Z. mobilis; or branched chain of alpha-keto acid decarboxylase kdcA in EC 4.1.1.72 of L. lactis, as described in publication WO 2011/031147, which has been incorporated herein by reference in its entirety. Examples of amino acid aminotransferase include those in EC

2.6.1, such as EC 2.6.1.21; EC 2.6.1.39; EC 2.6.1.42; and EC

2.6.1.67. Examples of amino transferases include those in EC 2.6.1, such as EC 2.6.1.11; EC 2.6.1.13; EC 2.6.1.18; EC 2.6.1.19; EC 2.6.1.48; and EC 2.6.1.62.

[00201] In one embodiment, a-keto pimelate can be contacted with one or more enzymes that catalyze the ketone reduction in a-hydroxypimelate (for example, a carbonyl reductase such as Ec 1.1.1.184), which can then be converted in a-hydroxypimelate CoA using an enzyme that catalyzes the transfer of CoA (for example, a CoA-

transferase described above). Ar-hydroxypimelate CoA can be converted to 2-heptene dioic acid CoA with an enzyme that catalyzes the dehydration of arhydroxypimelate-CoA (for example, a hydro-lyse classified under EC 4.2.1 such as EC 4.2.1.2, EC 4.2. 1.17; EC 4.2.1.18; EC 4.2.1.59 and EC

4.2.1.61). 2-hepetene dioic acid CoA can be converted to pyelic-CoA using an enzyme that catalyzes the enoate reduction of 2-heptene dioic acid CoA. The appropriate enoate reductases are described above.

[00202] In some embodiments, the invention provides a recombinant host cell comprising one or more enzymes necessary to convert D, L-diaminopimelate, preferably to pimelate.

1.5.8 Crotonato Route

[00203] A source of pimeloil-Coa, and finally a source of pimelate, is a biosynthetic route where the beginning of the carbon source is crotonate. That biosynthetic route is illustrated in figure 9. See, for example, Mouttaki, H., et al., “Applied Envtl. Microbiology ”, 73: 930-938 (2001). The crotonate is broken down into acetyl-Coa. It has been reported that two thirds of acetyl-CoA produced in the biosynthetic route shown in figure 9, is transformed into acetate. In some embodiments, the invention provides a recombinant host cell that is deficient in the enzymes that would convert acetyl-CoA to acetate (for example, a phosphate-acetyltransferase and acetate-kinase such as that of acetyl-CoA) instead of the conversion of acetoacetyl -Coa and subsequently into glutaconyl-CoA in a number of metabolic steps as shown in figure 9. Glutaconyl-CoA is subsequently converted to glutaryl-CoA. Glutaryl-Coa is an extended chain to form 3-oxopimelyl-CoA. After a reduction, dehydration, and a second reduction, pimelil-CoA is obtained.

[00204] In the biosynthetic route shown in figure 9, pimelyl-CoA is finally transformed into cyclohexane carboxylate. This biosynthetic route was observed in S. aciditrophicus. Thus, in some embodiments, the invention provides a recombinant host cell in which the genes encoding the enzymes that would be converted without pimeloyl-Coa to cyclhex-1-ene-1-carboxyl-CoA and finally to cyclohexane carboxylate, are "knocked out", so that the metabolic route ends in pimeloil-CoA.

1.5.9 Tetralin degradation route

[00205] In some embodiments, pyelic acid semialdehyde is produced using the tetralin (benzocyclohexane) degradation route from Sphingomonas and Corynebacterium spp. 7T-oxoheptanoic acid is produced as an intermediate is the tetralin degradation route in Sphingomonas “macrogolitabid. López Sánchez, A., et al., “Appl. Environ. Microbiol. ”, 76: 110-118 (2010). Tetralin is metabolized through the cleavage of both, the aromatic ring, resulting in pyruvate and pyelic acid semialdehyde. Tetraline, an organic solvent, is a complex starting material that is derived from naphthalene. Naphthalene is currently produced from coal tar ("coal tar"), but the biosynthetic routes for the synthesis of naphthalene are known to occur in termites, fungi and some types of plants. Chen. J. et al. "Nature", 392: 558-559 (1998); Daisy, B.H., et al., "Microbiology", 148: 3737-3741 (2002); and Azuma, H., et al., "Phytochemistry", 42: 999-1004 (1996). The pyelic acid semialdehyde can be converted to 7-amino heptanoic acid or 7-hydroxyheptanoic acid as discussed above.

1.11 Hexamethylenediamine from 2-oxopimelate

[00206] Some embodiments of the invention also provide a production method and Hexamethylenediamine from 2-oxopimelate comprising using recombinant host cells. 2-oxopimelate is a known intermediate in the coenzyme B biosynthesis route in methanogenic Archae.

[00207] Thus, the invention provides a recombinant host cell comprising: an enzyme capable of converting 2-oxopimelate to 2-aminopimelate (for example, an aminotransferase enzyme in EC 2.6.1.67), an enzyme capable of converting 2-aminopimelate to 2 -amino-7-oxoheptanoate (for example, a reductase enzyme in EC 1.4.1), an enzyme capable of converting 2-amino-7-oxoheptanoate to 2,7-diaminoheptanoate (for example, a l-aminotransferase enzyme in EC 2.6 .1), and an enzyme capable of converting 2,7-diaminoheptanoate to hexanethylenediamine (for example, a decarboxylase enzyme in EC 4.1.1). In some embodiments, the recombinant host cells, capable of producing Hexamethylenediamine comprise any or all of these enzymes. Embodiments of the invention also provide a Hexamethylene diamine production method comprising the use of these recombinant host cells.

[00208] In some embodiments, the invention provides a method for producing Hexamethylenediamine from renewable raw material, such as sugar, fatty acids, glycerol, and synthesis gas. In some embodiments, the non-naturally occurring host cell capable of producing Hexamethylenediamine comprises any or all of the enzymes required to convert renewable raw material to Hexamethylenediamine.

2.2 Recombinant host cells

[00209] This description characterizes recombinant host cells that recombinantly express one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more seventeen or more, eighteen or more, nineteen or more, twenty or more, or even more) enzymes used to produce a compound in one of the routes described here and in the methods using said host cells to produce di- or trifunctional C7 alkanes. For example, a host cell may include one or more (as above) exogenous nucleic acids encoding one or more (as above) of the following: an enzyme that catalyzes the reductive deamination of D, L-diaminopimelate or alpha-aminopimielate; an enzyme that catalyzes the enoate reduction of 2-heptenedioic acid to pyelic acid; an enzyme that catalyzes the reduction of carboxylic acid from pyelic acid to semialdehyde and pyelic acid, an enzyme that catalyzes the amination and semialdehyde of pyelic acid to 7-aminoheptanoic acid, an enzyme that catalyzes the hydrolysis of 7- acid starch aminoheptanoic to enantolactam, an enzyme that catalyzes the dehydrogenation of 7-aminopetanoic acid aldehyde to 7-aminoheptanal; an enzyme that catalyzes the transfer of an amino group to 7T-aminoheptanal to produce 1.7 diaminoheptane; an enzyme that catalyzes the transfer of CoA to 2-heptenedioic acid to produce 2-heptene diacid-CoA; an enzyme that catalyzes the hydrolysis of pimeloyl-Coa thioester to produce pimelic acid; an enzyme that catalyzes the reduction of carboxylic acid from pyelic acid to semialdehyde of pyelic acid; an enzyme that catalyzes the transfer of an amino group from the alpha-amino pimelate to produce the alpha-keto-pimelate; an enzyme that catalyzes the reduction and carbonyl from alpha-keto-pimelate to alpha-hydroxy-pimelate; an enzyme that catalyzes the transfer of CoA to alpha hydroxy pimelate to produce alpha hydroxypimelate CoA; an enzyme that catalyzes the reduction of alpha hydroxypimelate to 2-heptenedioic acid; an enzyme that catalyzes the elongation of the alpha-keto-pimelate alpha-keto acid chain to alpha-keto-suberate, an enzyme that catalyzes the transfer of an amino group to alpha-keto-suberate to produce alpha-aminosuberate, an enzyme which catalyzes the decarboxylation of alpha-keto acid from alpha-aminosuberate to 7-aminoheptanoic acid; an enzyme that catalyzes the decarboxylation of alpha-keto alpha-keto suberate acid to produce pyelic acid semialdehyde; an enzyme that catalyzes the reduction of the 6-amino-2-heptenedioic acid enoate to produce pyelic acid; or an enzyme that catalyzes the hydrolysis of the pimeloyl- [acp] thioester to pyelic acid. Recombinant host cells can contain any subgroup consisting of one or more (as above) nucleic acids encoding one or more of the enzymes listed above.

[00210] Host cells that are used typically have a number of properties: they can be easily genetically modified, are tolerant of the conditions used in the method of the invention, and grow in cell densities that are industrially useful.

[00211] Optionally, the host cell can be a single cell microorganism, or it can be a cell in a cell line. The host cell can be of an allele-wild genotype. In this example, the enzyme that is used to catalyze one or more steps in the method of the invention is naturally present in the host cell and is expressed at a level that has industrial use in the methods of the invention. In an alternative, the host cell has been genetically modified to express the enzyme on a level that has industrial use in the methods of the embodiments of the invention. The enzyme can originate from the cell in which it is expressed. In an alternative, the enzyme is derived from a different strain or cell species.

[00212] In an alternative, the host cell is a prokaryote. In another alternative, it is a eukaryotic cell. Typically, single cell microorganisms are used.

[00213] The term prokaryotic cell includes Gram positive and Gram negative bacteria. Examples of suitable Gram negative bacteria include those of the genus Escherichia such as Escherichia coli; of the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; of the genus Corynebacteria such as Corynebacterium glutanicum; of the genus Cupriavidus such as Cupriavidus necator or curpiavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; of the genus Delftia such as Delftia acidovorans; of the Bacillus genus such as Bacillus subtilis; the genus lactobacillus such as Lactobacillus delbrueckii; or of the lactococcus genus as

Lactococcus lactis.

[00214] Eukaryotic host cells include those from yeast and other fungi, as well as, for example, insects, mice, rats, primates, or human cells. Examples of suitable eukaryotic host cells include yeasts or fungi obtained from the genus Aspergillus such as Aspergillus niger, from the genus Saccharomyces such as Sacchromyces cerevisiae; of the genus Candida such as (CC. tropicalis, C. albicans, C. cloacae, C. guillermondii, CC. intrmedia, c. maltosa, C. parapsilosis, and C. zeylenoides, of the genus Pichia, as well as Pichia pastoris, of the genus yarrowia such as Yarrowia lipolytica; of the genus Issatchenkia such as Issathenka orientalis; of the genus Debaryomyces such as Debaryomyces hansenii; of the genus Arxula such as Arxula adenoinivorans; or of the genus Kluyveromyces such as Kluyveromyces lactis or of the genus Exophialia, Mucorhan, Trichoderma, , Cladophialophora, Paecilomyces, Scedosporium, and Ophiostoma.

[00215] Host cells can be provided in a variety of different ways. The cells can be cell debris. This means that the cells are grown in the cultures and removed from the culture medium before being used as a.biocatalyst. The cells can be used directly in the following growth, or they can be stored before use. Typical storage methods include freezing. Alternatively, the cells are lyophilized before use. In an additional alternative, the cells are cell growths. This means that the cells perform their biocatalytic action while being cultivated. If the substrate for a particular biocatalytic reaction cannot cross the cell membrane to be transformed by the host cells, then in an alternative, a crude lysate can be used. A crude lysate is the initial suspension of cell components produced after cell lysis. Cell lysis can be performed by any means, including chemical or mechanical. Additional methods of lysis will be apparent to those skilled in the art in view of their general knowledge and the teachings here. In another alternative, a clarified lysate can be used. A clarified lysate can be prepared by centrifuging the crude lysate for non-lysed cell pellets and other cell debris.

[00216] In some embodiments, substantially pure cultures of recombinant host cells are provided. As used here, a "substantially pure culture" of a recombinant cell is a culture of those cells in which less than about 40% (ie less than about: 35%, 30%, 25%, 20%, 15% , 10%, 5%, 2%, 1%, 0.5%; 0.25%; 0.1%; 0.01%; 0.001%; 0.0001%; or even less) of the total number of cells viable in culture are viable cells in addition to other recombinant cells, for example, bacteria, fungi (including yeasts), mycoplasmas, or protozoan cells. The term “about” in this context means that the relevant percentage can be 15% of the specified percentage above or below the specified percentages. So, for example, about 20% can be 17% to 23%. Said recombinant cell culture includes the cells and a growth, storage, or transport medium. The medium can be liquid, semi-solid (for example, gelatinous medium), or frozen. The culture includes cells growing in a liquid medium or in / on the semi-solid medium or being stored or transported in a storage or transport medium, including a freeze storage medium or transportation medium. Cultures are in a culture vessel or storage vessel or substrate (for example, a culture disc, flask, or tube, or a storage flask or tube).

[00217] In one embodiment, the recombinant cells of this description can be harvested from a fermentation process by conventional methods such as filtration or centrifugation and formulated in a dry pellet, or dry powder formulation, while maintaining high activity. The processes for production and a dry powder whose composition and recombinant cell showing one or more of the activities described here may include spray-drying, freeze-drying, dry fluidized bed, vacuum drum drying, or agglomeration and the like. The compositions can be a self-stable, dry biocatalyst composition suitable for the biosynthetic methods described here. Drying methods such as freeze-drying, fluid bed drying or a method employing spheronization extrusion / pelletizing followed by fluid bed drying can be particularly useful. The temperatures for these processes can be <100ºC, but typically <70ºC to maintain high residual activity and stereoselectivity. The dry powder formulation should have a water content of O -10% PpP / w, typically 2-5% w / w. Stabilizing additives such as salts (eg, KCl), sugars, proteins, and the like can be included to improve thermal tolerance or to improve the drying characteristics of recombinant cells in the drying process.

[00218] In some embodiments, partially or fully purified enzymes can be used instead of or in combination with recombinant hosts or recombinant lysates or non-recombinant cells. Fully or partially purified enzymes can be fully or partially lysed purified from any of the recombinant cells described herein or non-recombinant cells. Purification methods are well known in the art. Partial or complete purification has the advantage that the enzyme of interest is separated from other cellular components that can interfere with the reaction catalyzed by the enzyme (either by reaction with the substrate or by converting the intermediates or the desired product to an unwanted compound). Purification does not add additional steps to any biocatalyst preparation, however, and therefore, may not be appropriate in all examples. Determining the suitability of using partially or fully purified enzymes will be well within the understanding of those skilled in the art by following the teachings described here.

[00219] In some embodiments, one or more conversions in the methods of the invention will be performed by the biocatalyst under aerobic conditions. In an alternative, one or more of the conversions in the method of the invention will be carried out under anaerobic conditions. In a further alternative, some steps of the method of the invention will be performed under aerobic conditions and some steps will be performed under anaerobic conditions.

2.3 Modification of whole cell biocatalysts:

[00220] The biocatalyst used in the methods of the invention can be an unmodified host cell of the species in which the enzyme naturally occurs. Typically, however, it is necessary to genetically modify the host cell to produce an unnatural (i.e., recombinant or constructed) host cell.

2.4 Chromosomal modification of the host cell genome:

[00221] In some embodiments, the chromosome of the entire cell biocatalyst (i.e., the host cell) has been modified. This modification can be an insertion, a deletion or a substitution of a nucleic acid in the chromosome. Methods for effecting modifications for the cell chromosome are well known, for example, transposon mutagenesis, Cre-Lox-mediated recombination, REd lambda and RecET-mediated recombination.

[00222] The stable integration within the chromosome of a nucleic acid is advantageous because it allows the maintenance of the nucleic acid without the need for a selectable marker.

[00223] By performing repeated insertions, it is possible to stably integrate a number of different nucleic acids within a host cell biocatalyst chromosome. The average of stable integration that the host cell can be cultivated for more than five generations without loss of change. Generally, stable genetic changes include changes that persist for more than 10 generations, particularly stable changes include changes that persist for more than 10 generations, particularly stable changes will persist for more than about 25 generations, and more particularly,

stable genetic modifications will be greater than 50 generations, including indefinitely.

2.5 Epissomal modification of the host cell biocatalyst genome:

[00224] In some embodiments, the episomal composition of the host cell genome is modified. Through the episome it means any autonomously replicating element, for example, a plasmid (which can be linear or circular), a cosmid, an artificial yeast chromosome (YAC), etc. Epissomal elements often place a metabolic charge in their host cell and, consequently, in the absence of any active division mechanism to ensure that at least one copy of the episome is present in each of the daughters after cell division, it is necessary to include a selectable marker.

2.6 Sequences of introduced nucleic acid:

[00225] Nucleic acids introduced into the cell can comprise one or more (for example, two or more, three or more, four or more, five or more, six or more; seven or more; eight or more; nine or more ; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; fifteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more; or even more) of a number of elements. Typically, one of the elements encodes an enzyme that is used to produce di- or trifunctional C7 alkanes or precursors for said molecules. In some embodiments, the nucleic acids encode a protein that is not an enzyme that works in the method of the invention.

[00226] Typically, a promoter is operably linked to a nucleic acid. The choice of the promoter will depend on the intended application, and can easily be determined by the technician in the matter. For example, if an enzyme-catalyzed reaction can be harmful in a particular host cell, it is desirable to use a regulated or inducible promoter, so that gene expression can be returned or withdrawn if necessary. Alternatively, it may be preferred to have expression directed by either a weak or strong constitutive promoter to ensure that the enzyme is expressed at all stages of growth. Examples of promoters suitable for eukaryotic cell systems include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV), steroid-inducible promoter, and the murine leukemia virus Moloney (MMLV) promoter . Examples of yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH), galactokinase (GALI) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Other suitable promoters in yeast are known to those skilled in the art. Examples of promoters suitable for bacterial cell systems include, but are not limited to: T7, T3, SP6, lac and trp promoters.

[00227] If necessary to ensure expression, the introduced nucleic acid can also be operably linked to understand the 5 'untranslated (RTU), 3' UTR, enhancer and / or terminal regions, as required. Said elements will be known to those skilled in the art.

2.7 Expression of multiple enzymes in a host cell:

[00228] In some embodiments, a single strain of host cell is used to express more than one (e.g., two or more; three or more; four or more; belt or more; six or more; seven or more; eight or more; nine or more; ten or more; eleven or more; twelve or more; thirteen or more; fourteen or more; sixteen or more; seventeen or more; eighteen or more; nineteen or more; twenty or more or even more) of the enzymes used in the methods of the invention.

In this example, multiple enzymes can be encoded by the same nucleic acid introduced.

In an alternative, the enzymes can be encoded in separate nucleic acid fragments introduced.

Enzymes can also be expressed from a single promoter (for example, by arranging enzymes in the form of an operon). In an alternative, the enzymes can be expressed from multiple separate promoters.

In some instances, the multiple separate promoters can be induced by the same chemicals (for example, each of the multiple enzymes can be expressed from the yeast GAL promoter, thus meaning that each gene is inducible with galactose). Other suitable promoters are known in the art.

Alternatively, each of the genes encoding an enzyme used in the method of the invention is under the control of a different promoter.

Thus, different enzymes can be introduced individually through the use of different inducing compounds.

In another alternative, an intermediate approach is used, where a number of enzymes are under the control of the same promoter, and a number of enzymes are under the control of different promoters.

This alternative can be particularly advantageous when numerous enzyme routes have been generated in a host cell and it is desirable to control each member of a route according to the other members of the route, but to control each of the routes separately.

[00229] Additional considerations can be made regarding whether the multiple enzymes are expressed from the chromosome or from the plasmid. In the examples, where the enzyme is expressed from the plasmid, advantageously each plasmid comprises a different origin and / or a different selectable marker.

[00230] The expression of multiple enzymes in a single cell is advantageous because, if the co-expressed enzymes act directly after another in a reaction route, it guarantees that the upstream enzyme product is immediately available to be acted by the enzyme downstream. This avoids the need for both (1) purification of the intermediate and the adjustment of the second reaction vessel to perform the second reaction; Or (ii) the need for the product to be exported from the cell comprising the upstream enzyme within the culture medium where it can be acted on with a cell comprising the downstream enzyme.

2.8 Chaperone systems:

[00231] When a cell was built by expressing a protein under unnatural conditions (for example, when a protein that is native to a species is expressed at levels above the natural level, or in an alternative, when a protein starts from a different species is expressed in a host cell) in some instances, that the protein will not be expressed in an active form. Instead it can double incorrectly, and it accumulates as a non-functional aggregate

“Inclusion body”. In this example, cells used to express the protein can be subjected to genetic modification of additional expressed chaperone proteins that are either able to prevent protein mis-folding, or are able to redouble from the aggregate state . The inclusion of said chaperone protein is advantageous because it increases the amount of active protein per cell and, therefore, increases the complete efficiency of the method of the invention. The expressed chaperones may be a host cell chaperone protein. Alternatively, the chaperones may be of the same species / strain as the protein. Typical chaperone proteins for expression include members of the GroEL / GroES family, and members of the DnaJ / DnaK / GrpE family. Homologues of the E. coli archetype of the GroEL / GroES family, and DnaJ / DnaK / GrpE proteins have been identified in other prokaryotic species (see, for example), and eukaryotic homologues are also (GroEL and GroES corresponds to the eukaryotic proteins Hsp60 and Hspl0, and DnaJ, DnaK and GrpE corresponds to the eukaryotic proteins Hsp70, Hsp40 and Hsp24, respectively). These proteins have been identified in a number of yeast species (for example, Saccharomyces cerevisiae). The choice of chaperone proteins suitable for coexpression with an enzyme used in the method of the invention will be apparent to the skilled artisan, following the teachings described herein.

2.9 Metabolic construction of host cells:

[00232] Metabolic construction is the process of optimizing the parameters in a host cell in order to increase the capacity of a cell to produce a compound. The host cells used in the method of the present invention were optionally constructed to optimize the productivity of the difunctional C7 alkanes discussed above.

[00233] The metabolic construction to increase a cell's ability to produce a compound is mainly performed via two routes. The first is to optimize the enzymes in the production route of the desired product from the starting material. In a multiple enzyme route resulting in the production of a C7 alkane (as shown in the figures and described in the preceding sections), it is possible to determine the concentration of each intermediate in the route using techniques known to those skilled in the art (for example, two dimensional electrophoresis, the use of isotopically labeled precursors, and nuclear magnetic resonance (NMR) spectroscopy and, therefore, determine which of the enzyme conversions is the limit step of the step - this means that the step in the reaction scheme is decreased. This can be determined by observing an intensification of an intermediate, which indicates that the enzyme acting on this intermediate is limiting the total conversion rate. In this example, the rate at which this intermediate is reacted must therefore be increased. This can be done by the number of averages. First, the level of expression of the limiting enzyme can be increased. Optionally, this can be achieved by placing the gene encoding the enzyme under the control of a strong promoter, for example, the T7 promoter if the enzyme is being expressed in E. coli or the TEF promoter if the enzymes are being expressed in yeast. The second option is to increase the number of copies of the gene encoding the enzyme that is present in a cell, for example, by placing the gene in a multiple copy plasmid, or by incorporating multiple copies of the gene into the host cell's chromosome ( these copies can be incorporated at the same location on the chromosome or at different locations on the chromosome).

[00234] The production of difunctional C7 alkanes (as shown in the figures and described in the preceding sections) can also be increased by inactivating or reducing the activity of any enzymes that are able to bypass the substrate, any of the intermediates, or the product within a metabolic pathway other than assisting the method. Therefore, the activity of the enzyme can be decreased to increase the yield of the difunctional C7 alkanes. Thus, in some embodiments, the recombinant host cells described here may have a deficiency in one or more enzymes (for example, by deleting nucleic acid encoding an enzyme of interest or reducing nucleic acid expression) that are able to divide the starting material of the method of the invention, or any intermediate produced in the reaction route producing difunctional C7 alkanes, for a different, unwanted end product. In an alternative, the enzyme is not deleted, but instead has changed so that it can act against the substrate, intermediate, or product, at a rate that is less than the rate of the allele-wild enzyme. In the example, where the enzyme catalyzes a reversible reaction, then the enzyme must be changed so that it acts only in the desired direction of the reaction.

[00235] For example, in some embodiments, a recombinant host cell may have a deficiency in an enzyme capable of converting pimeloyl- [acp] to 7-keto-8-aminopelargonic acid (KAPA) or 6-carboxyhexanoyl-CoA ( pimeloyl-CoA) in KAPA (for example, a deletion or inactivation of BioF, the 7-keto-8-aminopelargonic acid synthase gene (EC 2.3.1.47).

[00236] In some embodiments, a recombinant host cell may have a deficiency in diaminopimelate decarboxylase, which creates a lysine auxotrophy. A lysine auxotrophy can be particularly useful for deregulating the carbon flow in D, L-diaminopimelate, the intermediate precursor to lysine. A lysine auxotrophy would require a batch fed fermentation.

2.10 Growth of the entire cell biocatalyst

[00237] In some embodiments of the invention, host cells are used, which are grown (i.e., divided) in time for the cells to perform the conversion in the method of the invention. In these embodiments, the cells are grown under conditions that optimize the production of the desired difunctional C7 alkane. Any of the recombinant host cells described here can be cultured to produce and / or secrete biosynthetic products. As used here, the term culture is equivalent to the fermenter and the bioreactor.

2.10.1 Medium:

[00238] In some examples, the carbon source used to grow will be provided in the form of a nutrient broth, for example, half Luria or half yeast extract. In other examples, a defined medium (that is, a medium in which the concentration of each component is known) suitable for the growth of the host cell can be used. In an alternative, the growth medium comprises glucose as a carbon source. In another alternative, the carbon source used by host cells in the medium is glucose, sucrose, xylose, fatty acids and glycerol.

2.10.2 Growth of multiple strains in the culture table:

[00239] In some embodiments, enzymes that catalyze conversion to the method of the invention are present in more than one cell strain / species, where those more than one cell strain / species are used simultaneously. In the example, where multiple strains / species are grown when conversion to the method of the invention is performed (that is, cells are co-cultured), then the strains / species that are chosen must be selected so that a strain does not be competent to other strains. This relationship can be obtained by introducing an artificial symbiosis within co-culture. That is to say, that two species / strains that are used are each auxotrophic for an essential nutrient, but for different nutrients. In addition, the other strain must be built to produce an excess of that nutrient so that both strains can survive together in a crop when the crop's growth medium does not understand the two essential nutrients. The appropriate selection and auxotrophies will be within the understanding of a person skilled in the art following the teachings of the invention.

2.10.3 Fermentation:

[00240] The culture conditions described here can be staggered and grown continuously to manufacture heptane-1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, T-hydroxyheptanal, 7 aminoheptanoic acid, heptamethylenediamine, 1,7 -heptandiol, 7 aminoheptanal, 7-aminohpetanol or enantolactam. Examples of growth procedures include, for example, batch feed fermentation and batch separation, batch feed fermentation and continuous separation, or continuous fermentation and continuous separation.

All of these processes are well known in the art. fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of di- and tri-functional C7 alkanes.

Generally, and as a continuous culture procedure, the continuous and / or near continuous production of heptane-1,7-dioic acid, 7-oxoheptanoic acid, T-hydroxyheptanoic acid, 7T-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7-aminoheptanal, 7-aminoheptanol, or enantolactam previously described includes culturing a recombinant host cell producing heptane-1,7-dioic acid, 7-oxoheptanoic acid, T7-hydroxyheptanoic acid, 7-hydroxyheptanal acid, 7-aminoheptanoic, heptamethylenediamine, 1,7-heptandiol, 7-aminoheptanal, 7-aminoheptanol or enantolactam described here in the presence of sufficient nutrients and medium to support and / or almost sustain growth in an exponential phase.

Continuous culture under such conditions can include, for example, 1 day, 2, 3, 4, 5, 6, or 7 days or more.

In addition, continuous culture can include 1 week, 2 3, 4, or 5 or more weeks and even many months.

Alternatively, recombinant cells can be cultured for hours, if appropriate, for a particular application.

It should be understood that continuous and / or close to continuous culture conditions can also include all time intervals between these exemplified periods.

It should be further understood that the cultivation time of the host cell of the invention is long enough to produce a sufficient amount of the product for a desired purpose.

[00241] The fermentation procedures are well known in the art. Briefly, the fermentation for the biosynthetic production of heptane-1,7-dioic acid, 7 oxoheptanoic acid, 7-hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptanoic acid, heptamethylenediamine, 1,7-heptandiol, 7-aminohpetanal, 7 -aminohpetanol or enantolactam previously described can be used in, for example, fermentation with batch feeding and batch separation; batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[00242] In addition, the above fermentation procedures using heptane-1,7-dioic acid, 7-oxoheptanoic acid, 7T-hydroxyheptanoic acid, 7T-hydroxyheptanal, 7 aminoheptaonic acid, heptamethylenediamine, 1,7-heptandiol, 7 aminoheptanal , 7-aminoheptanol or enantolactam producers described herein for continuous production of substantial amounts thereof, heptane-1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminoheptaóico acid, heptamethylenediamine, 1, 7-heptandiol, 7-aminoheptanal, 7T-aminoheptanol or enantolactam producers can also, for example, be substantially subjected to additional processes to convert the product into other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical or biocatalytic conversions to convert the product into other compounds, if desired.

2.11 Compositions of the invention

[00243] The invention also provides compositions comprising recombinant host cells according to the invention and heptane-1,7-dioic acid, 7-oxoheptanoic acid, 7-hydroxyheptanoic acid, 7-hydroxyheptanal, 7-aminopetanoic acid, heptamethylenediamine, 1 , 7-heptandiol, 7-aminoheptanal, 7T-aminoheptanol and enantolactam. The invention also provides a composition comprising a recombinant host cell according to the invention and a raw material. In some embodiments, the raw material is a renewable raw material, for example, where the raw material is selected from the group consisting of glucose, sucrose, xylose, fatty acids and glycerol. In some embodiments, the raw material is a polyaromatic hydrocarbon, for example, benzene, toluene or chiquimate (“shikimate”).

3.1 Examples:

[00244] Having so far described the invention in a generic way, it will be more easily understood with reference to the following examples, which are provided by way of illustration and are not intended to be limiting. It should be understood that various modifications and changes can be made here to those exemplary embodiments without departing from the spirit and scope of protection of the invention.

3.1.1 Conversion of 2,6-diaminopimelate to 2-amino-5,6-dehydro pimelic acid (6-amino-2-heptene dioic acid) and 2-aminoheptanedioic acid to 2-heptenedioic acid by ammonia liases ( EC 4.3.1.-) (See figure 10).

3.1.1.1. Selection of the target MAL enzyme genes:

[00245] In the identification of the route involved in the conversion of 2,6-diamino pyelic acid into pyelic acid, which is used in the production of nylon-7,7 and in the conversion of pyelic acid into 7-amino-heptanoic acid, target genes to from these routes were selected.

In particular, the target enzymes are selected from the family of ammonia lyases (EC 4.3.1), including ammonia lyases metilaspartate (MAL; EC 4.3.1.2), based on their broad substrate specificity, presence on a wide host range, ability to be cloned and expressed exogenously; and crystal structure available for rational design of protein construction.

The MAL genes obtained from Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae were selected.

The MAL enzymes encoded by C. amalonaticus (GeneBank ABOO05294; fragment 1641-2882878596..879060NM MN) and C. tetanomorphum (GeneBank S48141; fragment 756-1997) have significant dissimilarity (57% identity). The Aspergillus oryzae MAL gene (GeneBank XM 001827609) is evolutionarily divergent (44% identity with Citrobacter amalonactius MAL, 39% identity with Clostridium tetanomorphum MAL), which may show different catalytic properties and / or selectivity.

Another unusual ammonia lyase which is a potential enzyme for the reductive deamination of 2,6-diaminopimelate and 2-aminoheptanedioic acid, is ammonia lyase D-glucosaminate (EC 4.3.1.9) obtained from Pseudomonas fluorescens (accession number BAD69624). This is an unusual ammonia lyase that catalyzes a water, B-elimination: D-glucosaminato-2dehydro-3-deoxy-D-gluconate + NH;

oH oH Hom: Ho tm -. . - OH - ç i -, HO Ho o o

3.1.1.2 Cloning of expression vectors containing target genes:

[00246] The target MAL gene selected from Citrobacter amalonaticus, Clostridium tetanomorphum, and Aspergillus oryzae were then cloned into an IPTG inducible by a pET21-a main chain using inABLE technology to generate I4 (pEeET21-a with the MAL gene from Citrobacter amalonaticus), I5 (pET21-a with the MAL gene of Clostridium tetanomorphum), and IG6 (pET21-a with the MAL gene of Aspergillus oryzae). First, the gene and the DNA vector are divided into truncated parts using software analysis. The potential restriction enzyme, for example, EarI, sites in the selected MAL genes and in the pET21 vector were identified and disrupted by introduction mutations. The EarI restriction sites located within the coding sequences have been disrupted, for example, by incorporating a silent mutation, as to avoid altering the encoded protein sequence. The resulting DNA was free from the Earl sites, and was used in the input sequence for software analysis to design the corresponding truncated parts, flanked by Earl (ordered by DNA 2.0), long and short oligonucleotides (ordered through Sigma- Aldrich), and long and short part oligonucleotides (ordered through Sigma-Aldrich). The 5 'end of the part and the ligand oligonucleotides form phosphorylated by incubating the primer in the current T4-kinase under appropriate reaction conditions (6 UM oligo, 1 PNK buffer, 1 mM ATP, 10U T4 kinase, 5 mM DTT , 5% (w / v) PEG 8000), at 37ºC for 30 minutes, followed by inactivation of the enzyme at 65ºC for 20 minutes.

[00247] Then, the phosphorylated binder oligos and phosphorylated oligos were annealed by mixing equimolar amounts of each oligo (50 UL at 6 UM) and then heating the mixture to 65ºC in a thermocycler before gradually lowering the temperature below 20ºC to form oligos with parts of partial double strips and binder oligos with partial double strips having specific balances of 16 bp (melting temperature above 75ºC). The resulting ringed part oligos (POA) and ringed ligand oligos (LOA) were then diluted to a final concentration of 1 mM with TE buffer (tris-EDTA).

[00248] Then, the partial fragments / ligands corresponding to the selected genes (citrobacter amalonaticus, Clostridium tetanomorfphum and Aspergillus oryzae MAL) were generated through successive cycles of FarI digestion of the cloned truncated parts followed by the connection of their respective ringed oligo part (POAZ8, POA29, or POA3O) its truncated part (TP28, TP29 or TP30), and the ligated oligos linked from the vector's main chain (LOA31l). The fusion of the ligand part corresponding to the main chain of the vector was prepared by the ligation and its ringed oligos (POA31), its truncated part (TP3l1) and the ringed oligos ligands both from the genes (LOAZ8, LOAZ29 or LOA3O). Ten and twenty times the molar excess of LAO and POA compared to the truncated part were used in the gene and the vector reactions respectively in favor of the link between the truncated part and the oligonucleotides during each EarI digestion / binding cycle. The reactions were carried out in a total volume of 50UL and were incubated in a thermocycler (Eppendorf Mastercycler Gradient). EarI digestion / binding cycles were achieved by changing the temperature between 37ºC and 16ºC, which corresponds to the optimum temperature for FarI digestion and binding using T4 DNA ligase respectively. The samples were loaded onto a 0.7% agarose gel, and the binder part was purified from the residual main chain through separation on agarose gel and gel extraction (QIAGEN QIAquick Gel Extraction Kit). The expected size fragments were observed after agarose gel electrophoresis for the preparation of partial ligand fragments. The ligation of the digested fragment with the ringed oligonucleotides shows 3bp compatible with balanced results in the formation of a non-cleavable part / ligand fragment.

[00249] The partial gene / ligand fusions were then combined with their respective partial fusion / ligand vectors using a 2-part arrangement to construct the E. coli expression vector using the previously generated ligand parts. The gene and vector of the fusions of the linker parts were mixed together, in an equimolar amount (0.1 pmol each), which resulted in the expected DNA fragment arrangement due to the specific balance of complementarity formed in the ringed oligonucleotide and the oligonucleotide annular binder from each part. The reactions were incubated at room temperature for 30 minutes before the NEB10fB chemically competent high efficiency transformation of EF cells. coli using 2 ul of the 10 ul reaction array of competent cells. The transformed cells were plated on LB-Amp-agar and incubated at 37ºC overnight. Five hundred clones were obtained for each array. Two random clones were then harvested from each array and the corresponding vectors were isolated using the QIAGEN QIAprep Miniprep kit.

[00250] The restriction analysis was performed on the previously isolated vectors to confirm the construction the correct arrangement, for example, the arrangement for the Citrobacter amalonaticus gene within the main chain of pET21- a. the clones obtained from the Citrobacter amalonaticus gene array in the E.coli expression vector were analyzed using Pvul and BmgBI to identify the positive clones for insertion. The clones obtained from the Clostridum tetanomorphum gene array in the E. coli expression vector were analyzed using PstI and AlwNI (figure 30, table 10) to identify the positive clones for insertion. The clones obtained from the Aspergillus oryzae gene array within the E. coli expression vector were analyzed using SphI and EcoRV to identify the positive clones for insertion. Restriction products from each sample were analyzed using agarose gel electrophoresis. The expected band pattern was observed for both clones from each arrangement confirmed the constriction of E. coli expression vectors collected from the MAL gene of Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae. The arrangement of the 17 negative control vectors (pET21-a control) was performed using the partial fusion / Pet21-a ligand, partial ligand / pET21-a was performed as a negative control, and clones from the generation of the free gene construct negative controls were analyzed using XmnI and AlwNI. To further confirm the proper arrangement of the expression vectors, the partial junctions between the 3 '”end of the vector's main chain and the 5' end of the genes, as well as between the 3" end of the gene and the 5 'end of the main chain of the vector were sequenced from one of the two clones obtained from each array.

3.1.1.3 Expression of exogenous MAL genes in E. coli:

[00251] The E. coli constructs obtained previously were used to study the expression of the MAL genes of Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae in E. coli. The ventors (10 ng DNA from each array) were used to transform electro-competent E. coli BL21 cells (DE3), and transformation and samples were placed on LB-Amp-agar. Transformants were obtained after overnight incubation at 37ºC.

[00252] A single clone was harvested from each array on the plate, and was used to inoculate 5 ml of the LB-Amp medium as an initial culture. ODr; ow was measured after overnight incubation at 37ºC and 250 rpm. Two mL of each initial culture (approximately 4.5x10 '- 6.2x10' cells) was used to inoculate 100 mL of LB-Amp, and the cultures were incubated in a 500 mL flask with non-constant shaking at 37ºC and 250 rpm until an OD of 0.6 to 0.8 is achieved. Protein expression, which is controlled by the T7 promoter in pET21-a, was induced by the addition of 1 mM IPTG (final concentration) and the cultures were further incubated at 37ºC and 250 rpm. 1 mL of each sample was taken before induction, 4 hours after induction, and 24 hours after induction. Cell growth was verified by measuring OD'o. The remaining culture left the incubation after 24 hours if induction was transferred to a 50 mL Falcon tube, harvested by centrifugation at 5000 rpm for 10 minutes and the cell pellets stored at -20ºC.

[00253] Then, the samples at the time point in each array were processed for SDS-PAGE analysis. The samples were centrifuged at 13,000 rpm for 2 minutes before the supernatant was removed and the cells were lysed using the Bugbuster protein extraction reagent supplemented with lysozyme (15 mg / ml) and benzonase (3.4 U / UL). The lysis reactions were then centrifuged at 13000 rpm for 2 minutes, and the soluble fraction was transferred to a new tube and the insoluble fraction was resuspended in water. 20 UL of each fraction were mixed with 80 UuL of SDS sample buffer (SDS-load buffer, 9% DTT and water) and the mixture was heated in a heat block for 5 minutes at 95ºC. 10 UL of each sample were then loaded onto 4-20% SDS-PAGE tricene gel to analyze the protein content of the soluble and insoluble fractions. A protein band of the expected size (45 kDa) was clearly visible in the soluble fraction after IPTG induction. No corresponding proteins were observed in the negative control experiment suggesting that the expected 45 kKDa MAL protein Citrobacter amalonaticus and the 45 kDa MAL protein Clostridium tetanomorphum were successfully expressed as soluble proteins in E. coli from the expression vector constructs arranged . No significant protein was observed in the insoluble fraction of any sample after induction. The 45 kDa MAL protein from Aspergillus oryzae was also expressed in E. coli from the arranged expression vector construct, but it was expressed as an isolated protein (a 45 kDa band was visible in the insoluble fraction after induction) (See , Figure 11A and 11B).

[00254] In order to improve the solubility of the MAL protein, from Aspergillus oryzae, the induction temperature and / or the concentration of the inducer has been reduced, which in some cases has been shown to result in increased protein solubility due to a lower rate in synthesis of the protein and therefore greater efficiency in folding the protein. These parameters were reduced using the same expression and the induction procedure described above, but the SDS-PAGE analysis showed that the Aspergillus oryzae MAL protein will still be insoluble (a 45 kDa band was visible in the insoluble fraction after induction). A yeast expression vector containing the Aspergillus oryzae MAL protein can be used to express the protein in soluble form.

[00255] E. coli cultures expressing the MAL genes of Citrobacter amalonaticus our Clostridium tetanomorphum were scaled to increase the production of soluble proteins for use in enzymatic analysis. Cultures were grown, induced, and harvested as previously described. Briefly, a single clone was collected from each transformation plate previously prepared, and 20 ml of LB-Amp medium was inoculated as the initial culture. OD 'oo was measured after overnight incubation at 37ºC and 250 rpm. 16 ml of each initial culture (approximately 4.4x108 cells) were used to inoculate 800 ml of LB-Amp, and the cultures were incubated in a 2L non-uniform shaking flask at

37ºC and 250 rpm until an OD of 0.6 to 0.8 is achieved. Protein expression was induced by the addition of 1 mM IPTG (final concentration), and the cultures were further incubated at 37ºC and 250 rpm. After 24 hours of incubation after induction, the remaining culture was transferred to four 50 ml Falcon tubes, harvested by repetitive centrifugation at 5000 rpm for 10 minutes (4 rounds of centrifugation per tube; 50 ml of medium per round) and the cell pellets stored at -20ºC.

[00256] Lysis of the glass bead was used to break the cells for the collection of the MAL protein of Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae. Cell pellets from samples 24 hours after induction were resuspended in a lysis buffer consisting of 0.1 mM Tris, 1 mg / ml Pepstatin A and 200 mM PMSF protease inhibitors. Washed with 212-300 μm of glass beads (0.4 g per 1 ml of lysis buffer) were added to each tube, and the lysate in a parallel sonication water bath. 250 µl of samples for each of the 4 tubes were taken before sonication and after 5 minutes and 10 minutes of cell lysis. 250 ul of the samples at each point in time were taken to result in a total sample of 1 mL. The cell-free extracts were prepared by centrifugation for 10 minutes at 5000 rpm and transfer of the supernatant to new tubes. Insoluble samples were prepared after resuspending the isolated cell pellets after 10 minutes of sonication with an equivalent volume of water. The remaining pellets and the supernatant were stored at -20ºC. 20 µl of each pooled fraction was mixed with 80 µl of SDS sample buffer (SDS-

load, 9% DTT and water) and the mixture was heated in a heat block (“heatblock”) for 5 minutes at 95ºC. UL of each SDS sample was then loaded onto 4-20% SDS-PAGE tricene gel to analyze the protein content of the soluble and insoluble fractions. Protein bands were clearly detected in the samples, from the MAL expression of Citrobacter amalonaticus and Clostridium tetanomorphum.

3.1.1.4 Functionality of MAL expressed exogenously:

[00257] The activity of methyl aspartate ammonia lyase derived from Citrobacter amalonaticus, Clostridium tetanomorphum and Aspergillus oryzae was evaluated using strains I4, I5, and I6 in relation to beta-methyl aspartate, in the first place. In the subsequent studies, described here, the activity of these strains was evaluated in the context of different substrates, called, 2-aminopimelate, D, L-lysine and, finally, 2,6-diaminopimelic acid. A negative control of the bio-transformation of 2-aminopimelate with 1I17 was also conducted.

3.1.1.4.1 Classification of Biotransformation of strains I4, 15, and I6, in relation to beta-methyl-aspartate: NH, co, H and Ho. SE 4 NH HO, C

[00258] Six biotransformants were prepared in both 59 / L and 20 g / L, equivalent to the entire cell of the cell lysates of I4, I5 and I6. The concentration of the enzyme was calculated based on the whole cell equivalent which is the pelleted cell mass after centrifugation (net cell weight) divided by the volume of the buffer used to resuspend the cells during lysis. biotransformants were conducted in 3 ml volumes with 10 mM DL-threo-beta-methyl-aspartate.

[00259] The reactions were incubated at 30ºC in an orbital incubator and the samples removed for analysis by HPLC after 1, and 18 hours. The 1 ml aliquots were prepared for analysis by heat treatment to remove heat unstable proteins. The samples were subsequently centrifuged at 10,000 rpm for 2 minutes and filtered through a 2 micron filter. The analysis of the samples was performed by HPLC using a Phenomenex Rezex column with 5 mM sulfuric acid in mobile phase using RID and UV detection. Confirmation of lyase activity was confirmed by the detection of mesaconic acid in the samples. The concentration of mesaconic acid formed in each biotransformation was determined by measuring a standard external mesaconic acid obtained from Aldrich.

[00260] Mesaconic acid was detected in all biotransformations of strains I4 and 15, confirming that the functional expression of the lyase enzyme was present in these cell extracts. No activity was observed in strain I6 which is consistent with the results observed by the SDS-PAGE analysis of the expressed protein that showed the protein being expressed in insoluble form. The yield of mesaconic acid in all analyzed samples of strains I4 and I5 was -5 mM, which is consistent with the anticipated activity in 10 mM DL-threo-beta-methyl-aspartate, since the wild allele enzyme it is L-enantioselective. The complete conversion of strains I4 and I5 was observed after only 1 hour of incubation.

3.1.1.4.2 Classification of biotransformation of strains I4 and I5 in relation to 2-aminopimelate: NH, tee Neon TA Hoc NA> color + Nº

[00261] Eight biotransformations were prepared at varying levels of enzyme and substrate concentration (2 variables, 2 levels of stat design) to examine the activity of enzyme 14 and I5 in relation to 2-aminopimelate. Biotransformations were calculated in volumes of 3 ml with variable loads as indicated below: strain I4 5 g / L; concentration of 5 mM 2-aminopimelate T4 strain 5 g / L; concentration of 10 mM 2-aminopimelate T4 strain 20 g / L; concentration of 5 mM 2-aminopimelate strain I4 20 g / L; concentration of 10 mM 2-aminopimelate strain I5 5 g / L; concentration of 5 mM 2-aminopimelate strain I5 5 g / L; concentration of 10 mM 2-aminopimelate strain I5 20 g / L; concentration of 5 mM 2-aminopimelate strain 15 20 g / L; concentration of 10 mM 2-aminopimelate

[00262] Biotransformations were placed in a Falcon tube at 30ºC in a shaking incubator. The aliquots (1ml) were removed from the biotransformations after 7, 14 and 21 days. The prolonged incubation was operated to provide the best possible chance of observing lyase activity. The 1 ml aliquots were prepared for analysis by heat treatment to remove heat unstable proteins. The samples were subsequently centrifuged at 10,000 rpm for 2 minutes and filtered through a 0.2 micron filter. Sample analysis was performed by HPLC using a pre-column derivation method. This method comprises the formation and a chiral adduct with a Boc-cysteine and an ortho-phthaladehyde reagent to form a pair of diastereomeric derivatives of L- and D-2-aminopimelate. Diasteromers (representatives of the 2-aminopimelate enantiomers) can then be separated by HPLC using a C18 column and a 5 mM potassium phosphate buffer, pH 7.0 and an acetonitrile gradient at 338 nm. The peak areas for the L- and D-2-aminopimelate signals in each sample were recorded and the decrease in the L-2-aminopimelate pattern is observed and expressed as% relative to D-2-aminopimelate.

[00263] The data shows L-2-aminopimelate being degraded in all biotransformations. the increase in enantiomeric excess is consistent with the expected natural L-enantioselectivity for these lyase enzymes that preferentially act on L-beta-methyl-aspartate. Smaller substrate loads (5 mM) also showed greater .enantio- enrichment (ie, greater conversion of available substrate) compared to higher substrate loads that is consistent with anticipated lyase activity.

[00264] The presence of unreacted 2-aminopimelate was confirmed via mass spectrometry. M + H equal to 176.09 was observed in the sample and combined with the mass spectrum simulated for C; Hi3NO0s. In the total short of the mass spectrum, the product of enoic acid (M + H = 159.06) was not observed. However, when the MS accumulated ions for 20 seconds over a wide mass of 20 m / z, the isolation of the correct product was observed and combined with the simulated mass spectrum for C; H; i3NO.s.

[00265] A sample of the rac-2-aminopimelic acid starting material was also analyzed. Using the same method performed on this standard, the expected molecular ion for 2-aminopimelate was clearly observed. Meantime,

repeating the MS ion accumulated for 20 seconds over a wide mass of 20 m / z, the isolation (as was done in the previous biotransformation sample) revealed the presence of the enoic acid product as a mass fragment.

3.1.1.4.3 Biotransformation of the negative control of 2-aminopimelate with strain I17:

[00266] Four biotransformations were prepared with varying levels of cell layer and substrate to combine the levels used in the experiment described in 3.1.1.4.1. The biotransformations were carried out in reaction volumes of 3 ml as indicated below: strain I7 5 g / L; concentration of 5 mM 2-aminopimelate strain I7 5 g / L; concentration of 10 mM 2-aminopimelate strain I7 20 g / L; concentration of 5 mM 2-aminopimelate strain I7 20 g / L; concentration of 10 mM 2-aminopimelate

[00267] Biotransformations were incubated at 30ºC in an incubator with shaking and samples taken after 8, 13, and 21 days. The reaction analysis was performed by HPLC following the method described in the experiment above in 3.1.1.4.1.

[00268] The data show that there is some loss of L-enantiomer in strain Il7. However, the relative decrease in L-enantiomer is less than the equivalent biotransformation with strains I4 and I5, particularly when the second point in the 2-week incubation time has been compared. The trend shown below clearly shows that strains I4 and I5 in biotransformations have a higher level of consistency of L-2-aminopimelate degradation compared to strains of negative control I17 through the 2 x 2 statistical design experiment, examining the cell extract and substrate loads.

3.1.1.4.4 Classification of I4 and I5 CFE biotransformation with DL-lysine: NH, Ss HO, C NH,

[00269] Eight biotransformations were prepared in a similar way to that used to test lyase activity in relation to 2-aminopimelate. Strains lyase I4 and 15 were tested in varying concentrations of cell extract and DL-lysine substrate as follows: strain I4 5 g / L; concentration of 5 mM DL-lysine strain I4 5 g / L; concentration of 10 mM DL-lysine 20 g / L T4 strain; concentration of 5 mM DL-lysine strain I4 20 g / L; concentration of 2 DL-lysine 10 mM strain I5 5 g / L; concentration of 5 mM DL-lysine strain I5 5 g / L; concentration of 10 mM DL-lysine strain I5 20 g / L; concentration of 5 mM DL-lysine strain I5 20 9 / L; concentration of 10 mM DL-lysine

[00270] Biotransformations were placed in a Falcon tube at 30ºC in a shaking incubator. The aliquots (1ml) were removed from the biotransformations after 7, 14, and 21 days. The prolonged incubation was operated to provide the best possible chance of observing lyase activity. The 1 ml aliquots were prepared for analysis by heat treatment to remove heat-unstable proteins. The samples were subsequently centrifuged at 10,000 rpm for 2 minutes and filtered through a 0.2 micron filter. DL-lysine samples were analyzed using a chiral HPCL sample (Chirobiotic T2 Astec) to monitor the proportions of the enantiomer to determine lyase activity. The samples were removed from the biotransformations after 7, 14 and 21 days.

[00271] The results indicate loss of L-lysine in all biotransformations with 5 mM of lysine. Some variations in enantioselective action on DL-lysine were observed, where the concentration of L-lysine exceeded the concentration of DL-lysine. Due to the low UV absorbance (detect at 210 nm) and low signal to noise ratio of lysine in HPLC chromatography, the possibility of a false positive in the detection of L-lysine cannot be excluded from these results. However, in general, degradation and L-lysine from DL-lysine was observed through the eight biotransformations that is consistent with the expected stereo-selectivity of the potential lytic activity shown from the lyase functioning in strains I4 and 15.

[00272] In order to gather additional evidence to support the operation of lyase activity in these biotransformations, a sample was analyzed by MS to try to detect the desired enoic acid product in the sample. The MS signal corresponding to unreacted lysine (M + H = 176.09) was observed in the sample and combined with the simulated mass spectrum for C6H1aN2O2.

[00273] In the full mass spectrum scan, the product of enoic acid (M + H = 130.09) was not observed. However, as for amino-pyelic acid reactions, when ions accumulated in MS for 20 seconds over a wide mass isolation of 20 m / z, the correct product signal was observed and combined with the simulated mass spectrum for CÉsH11NO ?two.

[00274] A sample of the starting material of DL-lysine was also analyzed. Using the same MS method performed on this standard, the expected molecular ion for DL-lysine was clearly shown. However, the repetition of the accumulation of the MS ion for seconds during a large mass isolation of 20 m / z (as was done in the previous biotransformation sample revealed in the presence of the enoic acid product as a mass fragment.

3.1.1.4.5 Classification of Biotransformation of strains I4 and I5 CFE with meso-2,6-diaminopimelic acid: Ye and AA

NR An A ———— Hoc co, H + NH; HO, C co, H

[00275] Six biotransformations were prepared to test for lyase activity in relation to meso-2,6-diaminopimelic acid. Strains I4, I5 and I7 were tested with 5 mM meso-2,6-diaminopimelic acid, varying concentrations of cell extract were examined as follows: strain I4 5 g / L; concentration of 5 mM meso-2,6-diaminopimelic strain I4 5 g / L; concentration of 5 mM meso-2,6-diaminopimelic strain 15 20 g / L; concentration of 5 mM meso-2,6-diaminopimelic strain 15 20 g / L; concentration of 5 mM meso-2,6-diaminopimelic strain I17 5 g / L; concentration of 5 mM meso-2,6-diaminopimelic strain I17 5 g / L; concentration of 5 mM meso-2,6-diaminopimelic

[00276] Biotransformations were placed in a Falcon tube at 30ºC in a shaking incubator. The aliquots (1ml) were removed from the biotransformation after 4 days, further sample acquisitions are pending before HPLC analysis.

[00277] A sample acquired after 4 days was analyzed by MS. The analysis confirmed the presence of meso-2,6-diaminopimelic acid that matched the CiHiaN204 simulated mass signal.

[00278] The desired enoic acid signal was not observed in this mass spectrum in the biotransformation sample. However, when the ion accumulation was introduced for 0.56 seconds, then the corresponding enoic acid signal (M + H = 174.08) was observed in the sample.

3.1.2 Conversion of 2-amino-5,6-dehydro pyelic acid (6-amino-2-heptenedioic acid) to 2-amino-heptene dioic acid and 2-heptenedioic acid or its CoA ester to pyelic acid or its CoA- enoate reductase ester (EC 1.3.1.31) or enoyl-CoA dehydrogenases (EC 1.3.1.62) (See, figure 10A and 10B):

3.1.2.1 Selection of target enoate reductase:

[00279] During the identification of the route involved in the conversion of two unsaturated intermediates of pyelic acid, that is, 2-amino-5,6-dehydro pimelic acid and 2-heptenedioic acid, in their corresponding saturated compounds of 2-aminoheptanedioic acid and pyelic acid, the target enzyme were selected from the enoate reductase family (EC

1.3.1), such as EC 1.3.1.31, EC 1.3.1.8; EC 1.3.1.9; EC

1.3.1.10; EC 1.3.1.31; EC 1.3.1.38; EC 1.3.1.39; EC 1.3.1.44 or pimeloyl-CoA dehydrogenase in EC 1.3.1.62 such as PimC / PimD or ThnJ / ThnK. Enoate reductase can also be in EC 1.3, such as EC 1.3.8.1 or EC 1.3.99.3; EC 1.3.99.B10 or Syntrophys aciditrophicus of 2,3-didehydropyelyl-CoA reductase and homologues thereof, based on their different substrate specificity in relation to afb-unsaturated carboxylic acid and its stability (stero-complementarity of bioreduced dicarboxylic acid bioreduction) and dimethyl esters using enoate reductases - enzyme-based stereo-control and substrate: Stueckler et al., “Org. Lett.”, 2007, 9 - 5409- 5411).

[00280] In particular, the enoate reductase genes obtained from Bacillus subtillis (YqajM) (GeneBank D84432; fragment 242791- 243807) and Solanum lycopersicum (OPRIl and OPR3) (GeneBank NM 001247852, fragment 108-1238 and GeneBank NM 00124; -1284, respectively) were selected due to the fact that they were previously cloned and successfully expressed in structures of E. coli crystals, of the enzymes YajM and OPR3 have been reported, which enable the construction of the protein through rational design (O 1.3 a structure crystal of the YqjM flavoprotein reveals a new class of old yellow enzymes: Kitzing et al., “J. Biol. Chem.”, 2005: 280-27904- 27913; asymmetric bioreduction of activated alkenes using the 12-oxophtodienoatoclonate isoenzyme POR-l and POR-3 obtained from Lycopersicon esculentum (Tomato): A surprising change of stereo-preference: Hall et al., Angew, “Chem. Int. Ed.”, 2007, 46 - 3934-3937; 12-oxophytodienoate crystal structure reductase 3 from the tomato - author inhibition by dimerization: Breithaupt et al. PNAS 2006, 103, 14337-14342). In addition, XenA (EC 1.3.1.31) from Pseudomonas putida, TER (EC 1.3.1.44) obtained from Euglena gracilis, PECR and MECR obtained from Homo sapiens, acdA (EC

1.3.99.-) obtained from Pseudomonas putida, ACADS (EC 1.3.99.-) obtained from Bos Taurus and PimC / pimD (EC 1.3.1.62), obtained from Rhodopseudomonas palustres were selected for expression in E. coli and tested for reduction of 2-heptene-dioic acid and 2-heptenedioic acid-CoA.

BAcillus subtilis (YgjM) Fragment D84432 EO 242791-243807 Solanum lycopersicum (OPRI1) NM 001247852, [Fragment 1098-1238 Solanum lycopersicum (OPR3) NM 001246944; [The 94-1284 Pseudomonas putida, Xenobiotic AAFO2538.1 reductase A (XenA, 39.8 kDa, 3632AA) Euglena gracilis mitochondrial Trans-AAWG66853.1 2-enoyl-Coa reductase (TER, 43.8 kDa, 405 AA) Homo sapiens peroxisomal trans-2- NP 060911.2 enoyl-CoA reductase (PECR, 32.5 kDA, 303AA) Homo sapiens mitochondrial trans-2- NP 057095.2 enoyl-CoA reductase (MECR, 40.5 kDa, 373 AA) Pseudomonas putida acilenas-CoA N7 7 acdA, 41.6 kDa, 383AA) Bos taurus acyl-CoA dehydrogenase NP 001029573.1 (ACADS, 44.6 kDa, 412 AA) Rhodopseudomonas palustres pimeloil- NP 949051.1 CoA dehydrogenase (PimC, large subunit, 44.5 kDa Rhodes, 396AA, 396A marbles, pimeloil- NP 949050.1 CoA dehydrogenase (PimD, small subunit, 40.5 kDa, 3892AA)

[00281] Other enoate reductase that are potential candidates for reduction of the 2,3- double bond are those of the Clostridum species due to their broad substrate specificity (compounds —cquired synthesized by biocatalytic reductions: Simon et al., “Angew. Chem. Ed. ”, Engl. 1985 24 —- 539-553; properties and mechanical aspects of new discoveries of redox enzymes obtained from anaerobes suitable for bioconversions at preparatory scales: Simon et al.,“ Pure & Appl. Chem. ”, 1992, 64, 1181-1186). However, those enzymes were not cloned and expressed for the initial proof of concept due to their high sensitivity to oxygen (Enoato reductases from Clostridia - cloning, sequencing and expression: Rohdich et al., “J. Biol. Chem.”, 2001, 276, 5779-5787), but can be useful catalysts for the final construction route in an anaerobic host. In particular, the following enoate reductases should be useful for the reduction and double bonds in the target 2-enoyl C7 compounds:

[00282] Enr 2-enoate reductase (Y16137) EC 1.3.1.31 obtained from Clostridium klyyveri: all Clostridium kluyveri cells were observed to reduce a wide range of 2-enoates in the presence of molecular hydrogen. The substrates were: tiglic acid, acrylic acid, methacrylic acid, dimethylacrylic acid, (E) -pentenoic acid, (E) -hexenoic acid, sorbic acid, cinnamic acid- [19], (E) -2- butenoic acid and acid (E) -2-methyl-2-butenoic acid (E) - and (Z2) - 3-methyl-2-pentenoic acid, p-methoxy-cinnamic acid and p-nitro-cinnamic acid [Búhler, M., Giesel , H., Tischer, W. and Simon, H. (1980), "Ocurrence and the possible physiological role of 2-enoate reductases", FEBS Lett. 109, 244-246]. A partial sequence of the gene encoding the enzyme has been identified [Rohdich, F., Wiese, A., Feicht, R., Somon, H. and Bacher, A. (2001), Enoato Clostridia reductases, cloning, sequencing, and expression , “J. Biol. Chem. ”, 276, 5779-5787]. Once the C. kluyveri genome was sequenced, we looked at the entire gene sequence (YP 001394144.1, GENEBANK).

[00283] Enr 2-enoate reductase (Y09960) EC 1.3.1.31 obtained from C. tyrobutyricum (previously Clostridium sp. La 1): 2- enoate reductase was observed in Clostridium sp., Growing on (E) -butenoate and purified to homogeneity. Accepted

(E) -2-methylbutenoate and cinnamate [Elshahed MS, Bhupathiraju VK, Wofford NQ, Nanny MA, McInerney MF. (2001), Metabolism of Benzoate, cyclohex-1-ene carboxilate, and cyclhexene carboxilate by “Syntrophys aciditrophicus” strain SB in syntrophic association with H (2) -using microorganisms. Appl. Environ. Microbiol. 67: 1728-17380, (E) lime-formylamino-kinamate, and 4-methyl-2-pentenoate (Búhler, M., Geiesel, H., Tisher, W. and Simon, H. (1980), “Ocurrence and the possible physiological role of 2-enoate reductases. ”FEBS Lett. 109, 244-246]). the gene encoding enr reductase has been identified and the enzyme has been overexposed in E. coli under anaerobic conditions (Rodhich, F., Wiese, A., Feiht, R., Simon, H. and Bacher., A., ( 2001) - "Enoate reductase of Clostridia, Cloning, Sequencing, and Experssion - J. Biol. Chem.", 276, 5779-5787].

[00284] enr 2-enoate reductase (Y16136) EC 1.3.1.31 obtained from Moorella thermoacetica (previously Clostridium thermoaceticum): an anti-enoate reductase antiserum obtained from C. tyrobutyricum cross-reacting with the double bond in 2-heptenoedioic acids corresponding CoA esters.

[00285] The activity of pimeloyl-CoA dehydrogenase was observed in cell extracts of Syntrophys aciditrophicus grown in a co-culture with the strain Gll Desulfovibrio sp. With benzoate or in a pure crotonate culture and is involved in the benzoate degradation route (Elshahed MS, Bhupathiraju VK, Wofford NQ, Nannu MA, McInerney MJ, (2001) “Metabolism of Dbenzoate, cyclohex-l-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" strain SB in syntrophic association with H (2) -using microorganism. - Appl. Environ. Microbiol. ", 67: 1728-1738). It is believed that a route for the formation of cyclohexane carboxylate from crotonate involves the reduction and glutaconyl-CoA in glutaryl-CoA and 2,3-didehydropyelil-Coa in pimeloyl-CoA. The enzymes responsible for this activity have been identified, but the JS. Aciditrophicus, in a BLAST searcher using the non-decarboxylating Glutaryl-Coa dehydrogenases Desulfococcus multivorans (EC 1.3.99.B10) as an identified system of six sequences annotated as acyl-CoA dehydrogenases in Ss. aciditrophicus: YP 462067.1 (73% identity), YP 460269.1 (39%), YP 461117.1 (36%), YP 460428.1 (34%), YP 460268.1 (32%), and YP 463031.1 (28%). Wischgoll et al., (2009) compared the sequences of a number of glutariol-Co-enzyme A dehydrogenases and primary structure identified similarity between YP 462067.1 and the Desulfococcus multivorans gene which indicated that the trainer is also non-decarboxylating. Although the dehydrogenase reaction is the reverse of what is required, the proposed cyclohexane carboxylate formation route indicates that enoyl-CoA reduction occurs in this organism. Other hypothetical pimeloyl-CoA dehydrogenases seen in the databases include ThnJ (D9PTNO) and ThnK (D9PTM9) obtained from Shpingomonas macrogolitabid - observed in the gene group for tetralin degradation via beta-oxidation pathway, PimC (Q6N31IO0) and PimD ( Q6N3I11) of Rhodopseudoonas palustris and PimC (ASES10) and PimD (ASESO9) obtained from Bradyrhizobium sp., PImC / PimD were included in the selection for expression and biotransformation to evaluate the reduction of 2-heptenedioic acid and its CoA ester.

[00286] Other dehydrogenases useful for the invention include GDH-glutaryl-Coenzyme A dehydrogenase obtained from Desulfococcus multivorans, a non-decarboxylation glutaconyl-coenzyme A forming the glutaryl-coenzyme A dehydrogenase was characterized by the obligatorily anaerobic bacteria Desulfococcus multivorus , Demmer U., Warkentin E., Gúnther R., Boll M., Ermler U., (2010) “Structural basis for promoting and preventing decarboxylation in glutaryl-coenzyme A dehydrogenases. - Biochemistry ”, 49: 5350-5357]. The enzyme was overexpressed and its crystal structure was determined, which makes it a useful target for protein construction, to catalyze reversible oxidation and the reduction of C7 equivalents (2,3-didehydropyeloil-CoA and pimeloil-Coa) native C5 substrates glutacolonyl-CoA and glutaryl-CoA.

3.1.2.2 Cloning of expression vectors containing the target genes:

[00287] The selected target genes YqjM, OPRl, and OPR3 obtained from Bacillus subtilis and Solonum lycopersicum were then cloned within IPTG inducible by the main chain of pET21-a using 1inABLE technology, as described in detail in Section 3.1.1.2, to generate Il (the YqjM gene of Bacillus subtilis in pET21-a), I2 (the OPRI gene of Solanum lycopersicun in pET21-a) a and I3 (the OPR2 gene of Solanum lycopersicum in pET21-a).

[00288] Briefly, the potential EarI sites in the selected genes and the vector's main strand have been disrupted to prevent interference with the preparation of the partial / ligand fusion involving the Earl digestion / ligation cycles. No FarI site was observed in the YajM enoate reductase gene of Bacillus subtilis, but one and two EarI sites were detected in the OPR1 and OPR3 genes of Solanum lycopersicum, respectively, which was disrupted by the incorporation of silent mutation at the restriction site. The disruption of EarI 1 and 2 sites in the OPR3 gene sequence of Solanum lycopersicum resulted in an inadvertent site being created, which was only discovered after the sequences were synthesized. The residual EarI site was removed using Strategene's single site Quikchange mutagenesis kit. The forward and reverse primers were designed to disrupt the residual EarI site, and the primers were phosphorylated as described in section 3.1.1.2 prior to the Quikchange reaction. The reactions with Quikchange and negative control were prepared as follows: Reagent Change Quick control TP27 | negative Reaction buffer with turbo 10x Pfu Tr? ANTP solution Water in molecular biology grade 32 ul 33 ul Total Volume 50 ul 50 ul

[00289] The reactions were run in a thermocycler (95ºC, 0.5 minute; 35 cycles of 95ºC for 0.5 minute; 55ºC, 1 minute; 68ºC for 10 minutes; 68ºC for 1 minute; 4ºC waiting) and then treated with DpnI (1 UL) to digest the methylated parental DNA at 37ºC for 2 hours. Digestion reactions (3 UuL) were used to transform electrocomponent TOP10 E. coli cells, and transformation samples (100 UuL) were plated on LB-Kam-agar. Approximately 100 clones were obtained on the quick change reaction plate (“Quickchange”) compared to nine of the negative control plates after overnight incubation at 37ºC. Ten clones were removed from the plate, the corresponding plasmids were purified, and analyzed by EarI digestion. The absence of a 300 bp band on the agarose gel indicated that the EarI site located in the OPR3 gene was deleted. EarI digestion showed that several clones were positive for removal of the residual Earl site. DNA sequencing confirmed the clone sequence.

[00290] The vector of clone 3 (a positive clone for removal of EarI) was used to transform the electrocomponent TOP10 E. coli cells. The QIAGEN Hi-Speed Plasmid Purification Midi kit was used to prepare a large stock of DNA from the culture, and the DNA sample was subsequently concentrated using the QIAGEN PCR purification kit. The final DNA concentration and sample volume were 470.0 ng / Ul in 120 µl (conc. DNA 199.8 nM).

[00291] Three EarI sites were detected in the main chain of the pET21-a vector, which were disrupted by mutating the last guanidine base of the restriction site within a thymine (corresponding to the first cytosine within an adenine in the reverse complementary sequence) .

[00292] EarI free strings were then used as input strings in the partial design software to construct the corresponding truncated parts of the flanked by EarI sites, as discussed in Section

3.1.1.2. the truncated parts of YqjM synthesized from Bacillus subtilis (TP25), OPR1I from Solanum lycopersicum (TP26), and OPR3 from Solanum lycopersicum (TP27), were inserted into the DNAZ.0 vector pJ201. The truncated part vector pET21-a was completely synthesized with the introduction of a fragment including the chloramphenicol resistance gene to produce the TP31 vector.

[00293] Phosphorylation and subsequent annealing of inABLE oligonucleotides were performed as described in section 3.1.1.2.

[00294] Then, part of the ligation fusion was prepared by ligating the 5 'end of the truncated part with its corresponding oligo part of the ring fragment, and the 3 "end of the truncated part with the ligand oligo of the ring fragment, as previously described in section 3.1.1.2 The fusion of the binding part corresponding to the YqjM gene of Bacillus subtilis, OPRI of Solanum lycopersicum (TP26), and OPR3 of Solanum lycopersicum (TP27), were prepared by the bonding of their respective oligo ringed parts, their truncated parts , and the ligand-linked oligos from the vector's main chain The fusions of the ligand parts corresponding to the vector's main chain were prepared by the ligation of its ringed oligo parts, its truncated parts, and the ringed ligand oligos from its genes. A negative control of gene-free arrangement was also prepared by binding the oligo ringed part of the vector's main chain, its truncated part, and its ringed oligo ligand , resulting in a self-arrangement of the vector's main chain.

[00295] A 10- and 20-fold molar excess of the oligos and ligands to the truncated part was used in the reactions of the gene and the vector to favor the link between the truncated part and the oligonucleotides during each digestion with EarI / ligation cycle . The reactions with 50UL of EarI digestion / ligation were incubated in a thermocycler (Eppendorf Mastercycler Gradient), alternating the temperature between 37ºC and 16ºC. Upon completion of the cycles, samples were loaded onto a 0.7% agarose gel, and fragments of expected size were observed for the preparation of the part / ligand fusions, for example, a part / pET21-a ligand from YqjM from Bacillus subtilis, and a part / pET21-a OPR1 linker from Solanum lycopersicum as well as a 3 kb veotr backbone; or an OPR3 ligand from Solanum lycopersicum / part-pET21-a - 5.3 kg of Solanum lycopersicum OPR3 ligand / part-pET21-a - 1.2 kb as well as a 1.8 kg band corresponding to the main chain of the vector. The correct size of the bands was then cut from the gel, and the DNA was extracted from the gel using the QIAGEN QIAquick gel extraction kit. The DNA concentration varied from 14.4 ng / Ul to 49, ug / Ul in a total volume of 30 ul (conc. DNA ranged from 5.4 nM to 35.6 nM).

[00296] The combination of the fusion of the gene / ligand part with their respective fusion of the vector / ligand part was then performed through 2-part arrangements as described in the section

3.1.1.2. In summary, the gene part ligands and the vector part ligands were incubated at room temperature for 30 minutes before the high efficiency transformation with chemically competent E. coli OPR3 cells NEB10B using 2 ml of the arrangement reaction and 10 ul competent cells. The transformed cells were plated on LB-Amp-agar, and incubated at 37 ° C overnight. Five hundred clones were obtained for each array. One or two random clones were selected from each array, and the corresponding vectors were isolated using a QIAGEM QIAprep Miniprep kit.

[00297] The restriction was performed to confirm the construction of the vector, as described in section 3.1.1.2. In summary, the clones obtained from array 11 were analyzed using PstIn and HincII to identify the positive clones for the insertion of the Bacillus subtilis YajM gene into the E. coli pET21-1 expression vector, the clones obtained from the arrangement I3 were analyzed using BglII and Mlul. The restriction products were run on an agarose gel, and the expected band pattern was observed for the tested clones from each array, confirming the construction of E. coli pET21-a expression vectors harbored in the enoate reductase genes. from YqjM of Bacillus subtilis, OPRI from Solanum lycopersicum, and OPR3 from Solanum lycopersicum. In addition, the junctions of the parts between the 3 'end of the vector's main chain and the 5 "end of the genes as well as between the 3' end of the gene the 5 'end of the vector's main chain were sequenced to confirm the construction.

3.1.2.3 Expression of exogenous enoate reductase genes in E. coli:

[00298] Initially, the cloned enoate reductase genes were expressed in E. coli using IPMG ImM and an induction temperature of 37º, as described above in the section

3.1.1.3. In summary, OD600 was measured for each culture pre-induction and at various points in time after induction. The remaining culture left in pre-incubation after 24 hours was transferred to a 50 mL Falcon tube, harvested by centrifugation at 5000 rpm for 10 minutes, and the protein content of the samples was analyzed using SDS-PAGE. A large protein band of the expected size (35 kDa) was clearly visible in the soluble fraction of the enoate reductase expression study in Bacillus subtilis YqgjM after IPTG induction. A large band was visible in the insoluble fraction obtained from the expression of enoate reductase in Solanum lycopersicum OPRI1 after induction by IPTG, suggesting that the expected 42 kDa OPR1 protein expressed from 12 was insoluble in E. coli. A protein band of the expected size (44 kDa) was clearly detected in the soluble portion after IPTG induction of enoate reductase 13 in Solanum lycopersicum OPR3, as well as in the insoluble fraction, suggesting that the 44 kDA enoate reductase protein from Solanum lycopersicum OPR3 was expressed in E. coli as a partially soluble form.

[00299] Based on the reports of functional expression of the enanate reductase of Solanum lycopersicum OPR1 in E. coli optimized in the literature, the conditions for the IPTIG concentration (range of 0.1-1 mM IPTG) and the induction temperature (range of 16-37ºC) were optimized in an attempt to improve the solubility of Solanum lycopersicum OPR1 enoate reductase in E. coli (Strassner et al. JBC 1999, 274, p. 35067-35073; Hall et al., Angew, Chem. Int Ed. 2007, 46, p. 3934-3937). The same expression protocol and the same preparation of the SDS-PAGE sample as described in the section

3.1.1.3 was used for the solubility optimization experiments.

[00300] The samples at the time points of each array were then processed for and analyzed using the SDS-PAGE analysis, as described in section 3.1.1.3, which demonstrated that decreasing the induction temperature to 16ºC and the inductor concentration to 0 , 1 mM was effective to partially solubilize the enanate reductase from Solanum lycopersicum OPRI expressed in E. coli.

[00301] E. coli strains expressing the 3 enoate reductases were staggered in 800 ml cultures and evaluated for activity.

[00302] The other enoate reductases, XenA, TER, PECR, MECR, ACADS, PimC / PimD were expressed using standard techniques in E. coli, and purified on Histrap columns. Biotransformation reactions:

[00303] The reduction of the double bond of both substrates C6 (2-hexene dioic acid and 2,3-didehydroadipolyl-CoA) and C7 (2-heptenedioic acid and 2,3-didehydropyeloyl-CoaA) was evaluated.

[00304] The enzyme analyzes for enoyl-CoA reductase activity were based on published methods (Bergler H., et al., (1993). “Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli ”- The reactions were carried out in phosphate buffer (100 mM, pH 7.5), containing 300 UM of NADPH, NADH or FAD depending on the required enzyme cofactor, and the purified enzyme (25-55 Ug / ml). The reactions were initiated by the addition of the substrates, 2-heptene dioic or2,3-didehydropyloil-Coa. The reactions were incubated at 30ºC for 4 hours, and interrupted by the addition of 2M HCL (10% v / v). analyzed by LC-MS The controls were comprised of reaction mixtures without substrate and reaction mixtures with inactivated enzymes (boiled for 5 minutes).

[00305] The enzyme analysis for acyl-CoA dehydrogenase activity was based on published methods (Le W, Abbas AS, Sprecher H, Vockley J and Schulz H (2000)). The long chain of acyl-CoA dehydrogenase is the key enzyme in mitochondrial f-oxidation of unsaturated fatty acids. "Biochmica et Biophysica Acta", 1485: 121-128). The analyzes were carried out in 1 ml containing 50 mM of potassium phosphate buffer (pH 7.6), 36 UM dichlorophenolindopnenol, 0.3 mM

N-ethylmaleimide, 1.5 mM phenazine methosulfate, 60 µM acyl-Coa and enzyme (25-55 µg / ml). The reactions were initiated by the addition of fenzine methosulfate (Le et al., 2000).

[00306] The reactions were initiated by the addition of substrate (2,3-didehydropyeloyl-Coa or 2-heptenedioic acid) (60 UM). The reactions were carried out at 30ºC for 4 hours, and then stopped by adding 2 M HCl at 10% of the final volume. The products were analyzed by LC-MS. The controls used were reactions without substrate and reactions with boiled enzymes.

[00307] Results: ACADS, PimD, TER and XnA catalyzed the reduction of 2-hexenedioic acid to adipic acid (See, Figure 27A). MECR, PER TER and XenA catalyzed the reduction of trans-2,3-didehydroadipoyl-CoA in adipoyl-Coa (See, figure 27B). No formation of corresponding C7 substrates by these enzymes was observed under the tested reaction conditions, indicating that the enzymes had less activity for the C7 2-enoyl substrates and that the constructed enzyme would have required improved enzyme activity to be useful.

3.1.3 Conversion of pimeloil-Coa and pimeloil- [acp] to semialdehyde of pyelic acid by fatty acyl-CoA reductases (aldehyde forming FAR) (EC 1.2.1.50) or acyl- [acp] reductase (EC 1.2.1.80) ( See, figure 2):

3.1.3.1 - Selection of target enzymes

[00308] Fatty acyl-CoA reductase is involved in the synthesis of wax ester in plants and other higher eukaryotes for formation of the waxy cuticle. In Archaea and bacteria, these enzymes are involved in the biosynthesis of long chain alkanes, although their physiological function in these organisms is not yet clear. The formation of waxy esters in plants begins with the elongation of acyl-CoAs to form long-chain fatty acids (LFCA) (or fatty acyl-CoAs), which are then reduced via the decarbonylation route or reduction route and acyl-CoA (Cheesbrough and Kolattukudy, 1984).

[00309] Hydrocarbons are formed via the decarbonylation route. This route is initiated by a fatty acyl-CoA catalyst reaction to produce the aldehyde, followed by decarbonylation catalyzed by an aldehyde-decarboxylase to result in [Cn-1] -alkane (Cheesbrough eKolattukudy, 1984).

[00310] The acyl-CoA reduction route is also initiated by a reduction and LCFAs in aldehydes, followed by an additional reduction of aldehydes in an aldehyde reductase to produce still-linked primary alcohols (Millar et al., 1999) (Kolattukudy , 1971). Similar routes are taught to exist in prokaryotes (Schrirmer et al., 2010).

[00311] Both routes are initiated by the reduction of long-chain acyl-CoAs in aldehydes catalyzed by fatty acyl-CoA reductase (FAR; EC 1.2.150) in a reversible reaction. In the acyl-CoA reduction route, aldehydes are further reduced to alcohols. This can be achieved in either one or two stages, depending on the organism. The reduction in two steps is initiated using the catalysis reaction of acyl-CoA reductase, which releases the fatty aldehyde for a second reduction catalyzed by a grease aldehyde-reductase. One-step reduction is catalyzed by a bifunctional acyl-CoA reductase that reduces acyl-CoA directly in alcohol without releasing the aldehyde intermediate. Thus, fatty acyl-CoA reductase can be broadly divided into two types:

(1) acyl-CoA reductases with formation and alcohol; and (2) aldehyde generating acyl-CoA reductase. Examples of both types exist in prokaryotes and eukaryotes. Since the target is pyelic semialdehyde, the aldehyde-forming acyl-CoA reductase is the most promising candidate for reducing pimeloyl-CoA.

[00312] Preliminary literature and database searches revealed that most of the impressive acyl-CoA reductase studied or identified by sequence homology were alcohol-forming FARS in both eukaryotes and prokaryotes. However, one of the previous examples of an aldehyde-forming reductase came from Acinetobacter calcoaceticus - Acrl. Therefore, the primary sequence of this enzyme was used in the database search for other enzymes that generate aldehyde.

[00313] A general rule of thumb for acyl-CoA reductase appears to be that shorter proteins (295-350 aa) tend to be aldehyde-generating enzymes, while longer polypeptides (50+ aa) are alcohol producers. However, many short enzymes deposited in the databases have hypothetical notes or assumptions with no additional information beyond the sequence data and, for this reason, they have not been considered.

[00314] Candidates were chosen using the following criteria: (1) the enzyme must generate aldehyde only or must be in scope to eliminate the formation of alcohol by the constructed protein; (2) the existence of the protein must be evident, that is, the protein has been identified and purified or isolated in the cell fractions; (3) the enzyme was tested using acyl-CoA or acyl-ACP substrates; (4) the enzyme should not be part of a multiple enzyme complex unless activity has been demonstrated independently of the complex. Once the candidates were identified, additional bio-informatics and searches in the literature were carried out to provide additional data for the classification of the selections.

[00315] Table 1 shows the results of searches in databases carried out with respect to a known aldehyde-forming enzyme (Acrl). Each candidate was purified and evaluated in vitro. The candidates were classified and grouped according to their suitability. The highest rated enzymes (1-3) are considered the best candidates for further study, intermediate classified enzymes (4 and 5) are considered to be good candidates but may require further investigation and the lower rated enzymes (6) would require more efforts to obtain enzyme activity data, due to the absence of the protein sequence and the loss of a sequenced genome.

Table 1 Acyl-CoA aldehyde-forming candidate obtained from various organisms.

Organism name Substrate plus | No. From | Reduction | Protein / short level tested | amino- for evidence! Number of (No. Atoms of | acids | alcohol with carbon access) P94129 Acinetobacter 295 No Enzyme calcoaceticus activity YP 400611 | Synechococcus 341 No Activity elongatus PCC enzyme 7942 BAF92773 | Photobacterium 488 No Activity LuxC EC enzyme phosphoreum

1.2.1.50 YP 959769 | Marinobacter 661 Yes, but | Activity here VT8 may be possible enzyme truncate YP 959486 Marinobacter ND 513 Yes, but | Activity here VEL 8 may be | possible enzyme truncate AAB35106 Botryococcu 26 No Activity (partial) enzyme braunii * Level of evidence - activity of the enzyme means that the enzyme has been purified both heterologously and homologously, or that activity has been measured in crude extracts or other cell fractions.

[00316] Acinetobacter calcoaceticus Acrl (P94129) (Reiser, S., and Somerville, C., (1997): “Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester Synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzime The reductase ”- J. Bacteriol., 179, 2969-2975): An aldehyde-forming acyl-CoA reductase has been purified and contains 295 amino acids, with a molecular weight of 32.5 kDa. This enzyme was expressed in E. coli and cell-free extracts were shown to contain acyl-CoA reductase activity. Acyl-CoA substrates ranging from Cl12 to C24 in length were evaluated (but not the C- substrates added). The corresponding aldehyde was generated in all cases except for the Cl12 substrate. It would be worthwhile to further examine the substrate range of this enzyme and test its activity on shorter lengths of acyl-CoAs. Even if pimeloil-Coa is not a substrate, there is scope for direct evolution to extend the range of substrate. This enzyme was named directly in a patent related to the improved production of fatty acid derivatives (US 2011/0256599).

[00317] AAR obtained from Synechococcus elongatus PCC 7942 (YP 400611) (Schirmer, A., Rude, M.A., Li, X., Popova, E., and Del Cardayer, S.B. (2010)). “Microbial. Biosynthesis of Alkanes ”- Science, 329 - 559-562. This acyl-ACP reductase is in Cyanobacteria, S. elongatus, and is called AAr. The enzyme is researched for being involved in the biosynthesis of alkanes. It was expressed and purified in E. coli MG1I655. Both Coa and ACP derivatives are substrates. Thus, octadecanal is formed both from oleoil-CoA and oleoil-ACP without formation of fatty alcohol. However, oleoil-ACP was the preferred substrate, with a KM of 8 UM compared to 130 UM for oleoyl-Coa. The enzyme is dependent on NADPH and requires magnesium for activity. No other substrate has been tested. The selectivity for aldehyde formation and the activity with CoA derivatives makes this enzyme a good candidate to test for the reduction of pimeloyl-CoA. In addition, there are a number of orthologists of this gene in closely related organisms (Shirmer et al., 2010), although AAR is the best studied. The following patents are associated with this work (Shirmer et al., 2010): WO 2009/140695 and WO

2009/140696.

[00318] LuxC (BAF92773) of Photobacterium phosphoreum (Borylan, M., miyamoto, C., Wall, L., Graham, A., and Meighen, E. (1989). The Lux C, D and E genes encode luminescent operon of Vibrio fischeri for the reductase, transferase, and synthetase enzymes involved in the aldehyde biosynthesis. "Photochemistry and Photobiology", 49, 681-688; Lee, CY and Meighen, EA (1997). Cysteine-286 as the site of acylation of the Lux-specific for fatty acyl-Coa reductase. "Biochim. Biphys Acta", 1338, 215-222; Wang, X., and Kolattukudy, PE (1995). The solubilization and purification of fatty acyl-CoA reductase aldehyde from Botryococcus braunii green algae (FEBS Lett. 370, 15-18). LuxC is part of a multiple enzyme complex (LuxCDE) consisting of a fatty acyl-CoA reductase, fatty acyl synthetase and fatty acyl thioesterase, and is involved in bioluminescence. LuxC is a well-studied enzyme and has been cloned, expressed and purified in E. coli. The enzyme shows acyl-CoA reductase activity with the substrate c1l4, tetradecanoyl-CoA (myristoyl-CoA), which is reduced to the corresponding aldehyde, tetradecanal. No other substrate has been tested. Although LuxC is part of a complex of multiple subunits in vivo, the enzyme has been successfully purified from a wild allele organism and has been expressed and partially purified from E. coli. In both cases, LuxC was independently active from other subunits. This makes LuxC a good candidate for converting pimeloyl-Coa to semialdehyde of pyelic acid.

[00319] FACOAR obtained from Marinobacter aquaeolei VT8 (YP 959769.1) (Willis, R.M., Wahlen, B.D., Seefeldt, L.C.,

and Barney, B.M. (2011). Characterization of a fatty acyl-CoA reductase from Marinobater aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA in fatty alcohol. “Biochemistry”, 50 10550-10558): This fatty acyl-CoA reductase (referred to as FACOAR) was recently discovered in the range of proteobacteria - Marinobacter aquaeolei VT8. It is the first prokaryotic example of a bifunctional acyl-CoA reductase, catalyst for the reduction of acyl-CoA derivatives in alcohol. Crucially, it is only the enzyme that has been tested with substrates below C1l2, and has demonstrated activity with octanoyl-CoA in vitro (Willis et al., 2011). Although this enzyme generates alcohol from acyl-CoA, the reason for its inclusion is that there is clear evidence that it has a wide range of substrate from octanoyl-Coa (C8) to arachidonoil-CoA (C20). In addition, the C-terminal end demonstrates high sequence homology for the enzyme Acrl, so that the N-terminal region can be the alcohol forming domain. Therefore, it would be worthwhile to test the effect of removing the portion of the coding gene for the N-terminal region of the polypeptide, with the aid of the production of a functional aldehyde-generating enzyme. A potential problem with this approach is that the enzyme may be inactive or the aldehyde may not be released from the truncated enzyme complex. This enzyme has not been tested for activity against acyl-ACP derivatives.

[00320] FAR obtained Marinobacter aquaeolei VT8 (YP 959486) (Hofvander, P., Doan, T.T.P., and Hamberg, M. (2011)). A prokaryotic acyl-CoA reductase that performs the reduction and fatty acyl-CoA in fatty alcohol. “FEBS Letters”, 585 - 3538- 3543: This fatty acyl-CoA reductase (called FAR) was originally identified as a fatty aldehyde-reductase (Wahlen, BD, Oswald, WS, Seefeldt, LC, and Barney, BM (2009) ). Purification, characterization, and the production of potential bacterial wax participate in the NADPH-dependent fatty aldehyde reductase, obtained from Marinobacter aquaeolei VTS8. “Appl. Environ. Microbiol. ”, 75, 2758 - 2764, but has been shown to have acyl-CoA reductase activity (Hofvander, P., Doan, TTP, and Hamberg, M. (2011). A prokaryotic acyl-CoA that accomplishes the reduction of fatty acyl-Coa in fatty alcohol. “FEBS Letters”, 585, 3538-3543). It is very similar to the fatty acyl-CoA reductase described above, but it is also known to act on acyl-ACP derivatives. Activity was observed with Cl6-CoA through C20-Coa, but was not tested with shorter substrates. The enzyme is 513 amino acids long and has the same C-terminal domain associated with the production of aldehyde like Acrl. It is possible that this enzyme could also be truncated, which would mean cleavage of the N-terminal region as previously described. It is not clear whether or not FAR and FAcoR are entirely different enzymes, since two separate research groups have studied them.

[00321] Aldehyde-producing Acyl-CoA reductase obtained from Botryococus braunii (AAB35106) (Wang, X, and Kolattukudy, P.E. (1995)). “Solubilization and purification of aldehyde- generating fatty acyl-Coa reductase from green algae Botryococcus braunii. FEBS Lett ”, 370, 15-18): This enzyme is an aldehyde-producing acyl-CoA reductase obtained from a microalgae, Botryococcus braunii. The enzyme was purified from the allele-wild organism and evaluated using labeled activated fatty acid, [1-14C] -palmitoyl-CoA. The enzyme has been shown to produce the corresponding aldehyde and was dependent on NADH. The substrate range has not been studied and this would need to be addressed. In addition, the protein needs to be cloned, expressed and purified in E. coli. Accession number AAB35106 refers to the partial sequence of 26 N-terminal amino acids obtained from the purified enzyme. The complete protein sequence has not been deposited in the UNIPROT, BRENDA, or NCBI databases. Genome sequencing is on the way (http://www.jgi.doe.gov/). It may be in the scope to isolate the gene using primers for the N-terminal sequence, but the general loss of information regarding the enzyme can complicate its exploration for metabolic construction.

[00322] Thus, a number of acyl-CoA reductase have been identified, which are suitable candidates to be tested for the reduction of pimeloyl-CoA in pyelic semialdehyde. The enzyme obtained from Acinetobacter calcoaceticus, Acrl, seems to be the best candidate, since it was better characterized in the enzyme identified even though there is no information on the activity with substrates with short-chain C-few. If the initial tests are negative, the enzyme could still be a good target for targeted evolution to obtain the reduction of pimeloil-coa.

[00323] AAR obtained from S. elongatus and its orthologists, as well as LuxC are also promising candidates. Although these enzymes operate on relatively long-chain fatty acyl-CoA substrates, they have not been tested with short-chain substrates. Again, directed evolution can provide a promising route to obtain the desired substrate selectivity if the initial tests are negative.

[00324] The two enzymes of M. aquaeolei VT8 can also be promising, although some proteins constructed probably require the prevention of oxidation of the aldehyde. These enzymes are suitable candidates because they are known to act on shorter chain acyl-CoAs and all identified fatty acyl-CoA reductases. The gene encoding the enzyme from B. braunii has not been identified. This makes it the most difficult enzyme to work with, but it could be investigated if other options are unsuccessful. Similar enzymes are also known in higher plants (for example, Pisum sativum) and provide additional options for testing, but, again, are not well characterized.

[00325] Thus, the six enzymes above were selected because they are reported in the literature, and because they have been prepared, purified (partially or totally), both recombinantly and naturally in cellular fractions and evaluated in vitro. Its nucleotide and primary amino acid sequences are deposited in the main protein databases BRENDA and UNIPROT, except for the enzyme Botyrococcus braunii. The results from the studies were published in peer-reviewed journals. The tested substrate ranges from octanoil-CoA (C8) to arachinadoil-CoA (C20) and also included acyl-ACP as a substrate in the case of Synechococcus elongates AAR. The corresponding aldehydes are formed as products. However, currently, none of the enzymes has been tested with pimeloyl-CoA or any other number of CC substrates, presumably because the metabolism of these substrates is assumed, invariably, for having the same pattern followed, for those of substrates with the same number C and, so far, there is no reason to retest these substrates. Therefore, it is highly likely that the enzymes will catalyze the reduction of pimeloyl-CoA. Even if pimeloil-Coa is not a substrate, the enzymes would provide a good starting point for evolution towards or building the target protein.

[00326] Other candidates among the long-chain fatty acyl-CoA reductase in EC 1.2.1.50 for the conversion of pimeloyl-CoA to pyelic acid semialdehyde include, but are not limited to, reductases obtained from the following organisms: Pisum sativum ( Vioque et al., 1997. Resolution and purification of an aldenyde-generating and an alcohol- generating fatty acyl-CoA reductase from Pea leaves (Pisum sativum L.), Archives of Biochemistry and Biophysics, 340 (1), 64-72) ; Vibrio fischeri (P12748) (Boylan et al., 1985). Functional identification of fatty acid reductase components, encoded in the luminescence operon of Vibrio fischeri, “Journal of Bacteriology”, 163 (3), 1186-1 190; Brassica oleracea and B. napus (039342) (Kolattukudy, 1971. "Enzymatic synthesis of fatty alcohols in Brassica oleracea". "Archives of Biochemistry and Biophysics", 142 (2), 701 -709); Clostridium butyricum (Day et al., 1978). “Partial purification and properties of acyl-CoA reductase from Clostridium butyricum, Archives of Biochemistry and Biophysics”, 190 (1), 322-331; Anas. platyrhynchos (Ishige, T .; Tani, A .; Takabe, K .; Kawasaki, K .; Sakai, Y .; Kato, N. “Wax Ester Production from n-Alkanes by Acinetobacter sp. Strain M-1: Ultrastructure of Cellular Inclusions and Role of Acyl Coenzyme A Reductase, Appl Environ Microbiol 2002, 68, 1 192-1 195); . Arabidopsis thaliana (Doan, T. T. P .; Carlsson, A. S .; Hamberg, M .; Billow, L .; Stymne, S .; Olsson,

"P. Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli ”. Journal of plant physiology 2009, 166, 787-796; Hooks, M. A .; Kellas, F .; Graham, IA "Long-chain acyl-CoA oxidases of Arabidopsis". Plant J. 1999, 20, 1-13); Homo sapiens, R. P .; Wang, Y .; Mohsen, A.-W .; He, M .; Vockley, J .; Kim, J.-J. P. "Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA".

[00327] From these aldehyde-forming dehydrogenases in EC 1.2.1.50 described above, LuxC, the NADPH-specific cil-CoA reductase obtained from Photobacterium phosphoreum (accession number BAF92773.1) was chosen as an example to demonstrate the formation of semialdehyde of pyelic acid from pimeloyl-CoA. Other dehydrogenases of interest are found in EC.1.2.1.76 and EC 1.2.1.10. For example, Clostridium kluyvery DSM555 has a CoA-dependent semialdehyde succinate dehydrogenase (SucD, EC 1.2.1.76 accession number AAA92347), as well as 2 other aldehyde-dehydrogenases (accession number EDK 34221.1) and an acetaldehyde-dehydrogenase (EC 1.2 .1.10, accession number EDK33116) that can be used to convert pimloyl-CoA to pyelic acid semialdehyde. An additional enzyme in EC

1.2.1.10, PduB, a CoA-dependent propionaldehyde dehydrogenase obtained from Propionibacterium freudenreichii subspecies Shermanii (accession number D7GD28) was selected.

[00328] These 5 selected enzymes were cloned into the vector pET151 / Topo and expressed in E. coli BL21 (DE3) or Rosetta2 (DE3) using standard techniques. The activity was confirmed by monitoring the reaction at 340 nm for the disappearance of NADH or NADPH with acetyl-CoA as a substrate, as well as with hexanoyl-CoA. The enzyme SucD of Clostridium kluyveri was obtained in the insoluble fraction and the activity cannot be detected in the soluble fraction, since the breaking of the cell suspension of Clostridium kluyveri was used in the subsequent analysis of biotransformation.

[00329] Biotransformation to evaluate the activity of enzymes with pimeloyl-CoA with purified enzymes (Histrap columns) produced in E. coli were performed under aerobic conditions. A total reaction volume of 250 UuL made of DTT (5 mM), MgsSO4 (5 mM), NADH (1.2 mM), adipoyl-CoA or pimeloyl-CoA (1 mM) in Tris buffer (50 mM, pH 7 , 5) was prepared in a 96 well plate. The reaction was initiated by adding 10 to 15 ul of the soluble fraction of the proteins prepared in E. coli. Each reaction was done in triplicate. A “control buffer” was prepared by replacing the enzyme preparation with Tris (50 mM, pH 7.5). This control was prepared in triplicate. A "control enzyme" was prepared using the mixture described above, but replacing hexanoyl-Coa with water for each enzyme preparation. This control was done in duplicate.

[00330] All mixtures ("Analysis", "Control Buffer", "Control Enzyme") were prepared in parallel and were incubated in a 96-well plate overnight at 37ºC or 32ºC under 200 rpm shaking before being spun spinned down. Supernatants were added inside a 96 well plate with LC / MS for LC / MS analysis.

[00331] Biotransformations for the evaluation regarding the reduction of pimeloyl-CoA in semialdehyde of pyelic acid using fractions of Clostridium kluyveri enzyme, were carried out as follows: Culture of Clostridunm kKkluyveri and preparation of the enzymatic fraction:

[00332] 2 strains of C. kluyveri (DSM555 and DSM563) were grown in flask in ethanol (20 mL / L of culture) and succinate (5 g9 / L) in 40 mL of modified DSM-52 medium containing: K2HPO4 (0 , 31 g9g / L), KH2PO4 (0.23 g / L), NH4Cl (0.25 9 / L), MgSO04x7H20 (0.20 g / L), 70 mg / L of ZnCl2; 100 mg / L of MnCl2x4H20; 6 mg / L of H3BO3; 190 mg / L of Cocl2x6H20; 2 mg / L Cucl2x2H20; 24 mg / L of NiCl2x6H20; 36 mg / L Na2MoO4x2H20), selenite-tungstate solution (1 mL) (0.5 gg / L NaOH, 3 mg / L Na2SeO3x5H20; 4 mg / L Na2WO0O4x2H20), yeast extract (1 g / L ), resazurin 90.5 g9 / L), NaHCO3 (2.5 9 // L), seven vitamin solutions (1 mL) (100 mg / L vitamin Bl2; 80 mg / L p-aminoenoic acid; 20 mg / L of D (+) - biotin; 200 mg / L of nicotinic acid; 100 mg / L of calcium pantothenate; 300 mg / L of pyridoxine hydrochloride; 200 mg / L of thiamine-HClx2H20); cysteine-HClxH20 (0.25 g / L), Na2Sx9H20 (0.25 g / L). The medium was prepared strictly anaerobic.

[00333] After 4-5 days of growth at 30ºC, the culture was harvested and the pellets weighed. The pellet was lysed using the yeast extraction reagent Y-perO directly for 1 hour with N, gas. All procedures were performed in an anaerobic cabin. The soluble fraction obtained from the cultures of C. kluyveri was used in the exemplary reactions.

[00334] Biotransformation reactions with fractions of enzyme from the C. kluyveri strains were carried out in an anaerobic cabin as follows: the reactions were carried out in Tris buffer (50 mM, pH 7.5), DTT (5mM), MgSO4 ( 5 to 10 mM)

NADH (1 mM), NADPH (1 mM), adipoyl-A coenzyme or pimeloyl-A coenzyme (0.5 to 1 mM) in a total volume of 200 UL. The reaction was initiated by the addition of 15 UuL of the enzyme preparation (soluble or insoluble fraction). These reactions were done in triplicate. A "control buffer" was prepared to replace the enzymatic preparation with Tris (50 mM, pH 7.5). This control was prepared in triplicate. All mixtures ("Evaluation", "control buffer") were prepared in parallel and were incubated overnight at 30 ° C (or 22 ° C for P. phosphoreum) with shaking at 200 rpm before being turned down. Supernatants were added to an LC / MS 96-well plate for LC / MS analysis.

[00335] Detection of adipic- (C6) and pyelic acid (C7) semialdehyde was carried out using a mass-determined Q-TOF coupling with the infinite LC 1290 with an HTC / HTS (Agilent) autosampler. A Synergi-2.5u FusionRP column (Phenomenex, Ref: 00B-4423-BO) was used for analysis. The authenticity pattern of semialdehyde C6- and C7- was detected in the negative form using ultrapure water supplemented with 0.1% (v / v) formic acid and acetonitrile supplemented with 5% (v / v) ultrapure water as a buffer. running. The method used to detect semialdehyde authenticity patterns was used to analyze the reactions.

[00336] Of the enzymes expressed in E. coli and obtained in the soluble fraction that were evaluated under aerobic conditions, all enzymes showed activity detectable with acetyl-CoA and hexanoyl-CoA as determined spectrophotometrically. However, only CoA-dependent propionaldehyde dehydrogenase obtained from Propionibacterium freudenreichii subspecies Shermanii (accession number D7GD28) produced pyelic acid semialdehyde from pimeloyl-CoA as determined by LC-MS, despite the fact that only a small fraction of the enzyme was obtained in the soluble fraction. C. kluyveri enzymes are known to be sensitive to oxygen, so the loss of activity observed by the 3 C. kluyveri enzymes could be due to the evaluation conditions. Thus, the cellular fractions of C. Kluyveri, produced under anaerobic conditions with succinate as an inducer in the medium, and evaluated under anaerobic conditions in native strains, were used to determine the activity of SucD (EC 1.2.1.76). The enzyme fractions of both strains of C. kluyveri (DSM 555 and DSM563) used, produced detectable amounts of pyelic acid semialdehyde from pimeloyl-Coa as analyzed by LC-MS.

3.1.4 - Conversion of pyelic acid to pyelic acid semialdehyde through carboxylic acid reductases (EC

1.2.99.-):

3.1.4.1 Selection, cloning, and expression of target carboxylic acid reductases.

[00337] The target enzymes for the reduction of pyelic acid to pyelic acid semialdehyde were selected from the family of carboxylic acid reductases (EC 1.2.99.-, such as EC 1.2.99.6). In particular, Nocardia carboxylic acid reductase is a monomeric enzyme that participates in a large substrate specificity in relation to aromatic substrates and has been successfully cloned and expressed in E. coli (Nocardia sp., Carboxylic acid reductase: cloning, expression and characterization of a new aldehyde oxidoreductase family: He et al., “Appl. Eniron.

Microbiol. ”, 2004, 70, 1874-1881). The enzyme was therefore a good candidate for the start of classification to monitor the conversion of pyelic acid to pyelic acid semialdehyde.

[00338] The Nocardia Sp. (CAR) carboxylic acid reductase gene (GeneBank AY495695) was expressed in E. coli BL21 along with MNocardia phospopanthete transferase (PPTase) and used as a cell-free extract followed by cell lysis and centrifugation to remove the insoluble fraction. (See Venkitasubramanian, P .; Daniels, L .; Rosazza, J.P.N. Reduction of carboxylic acids in aldehyde oxidoreductase by Nocardia requiring a phospopanteteinylated enzyme. “J. Biol. Chem.”, 2007, 282, 478-485).

[00339] The expression study was then started using standard conditions (1 mM IPTG, 37ºC pre- and post-induction temperatures) and was expressed in E. coli under IPTG induction according to the previously reported protocol, and scaled to produce enough protein for activity analysis.

3.1.4.3 - Evaluation of the activity:

[00340] The evaluation of activity was based on the spectrophotometric detection of CAr activity in relation to sodium benzoate as the positive control and sodium pimelate. The conditions for the evaluation were taken from “Applied and Environmental Microbiology”, 2004, 70, p1874-1881.

[00341] Each evaluation comprising the final concentration of 50 mM of the substrate (100 UL), 100 mM of magnesium chloride (100 µl), 50 mM of trihydrochloric acid, 1 mM of ethylene diaminetetraacetic acid, 1 mM of added dithiothreitol together with a stock solution (200 ul), 10 mM adenosine triphosphate (100 ul), 1.5 mM nicotinamide-dinucleotide hydrate (reduced) (100 1ul) and the biocatalyst, which was loaded as a free extract cell to result in a final concentration of 4.3 g / L, the entire cell equivalent. The reactions were carried out at room temperature (-20ºC) in a 2 mL vat at pH 7.5. Each evaluation was first loaded with magnesium chloride (100 µl), 50 mM tris hydrochloric acid, 1 mM ethylene diaminetetraacetic acid, 1 mM dithiothreitol (200 µl), 10 mM adenosine tri-phosphate (100 µl) and 1.5 mM nicotinamide-dinucleotide phosphate hydrate (reduced (100 μl) and absorbance measurements recorded in thirty seconds (30 s) from zero time interval (Os) to 2 minutes (120 s). The biocatalyst was then carergadoe and the absorbance recorded in thirty seconds (30 s) interval from time zero (Os) to two minutes (120 s) .Finally, the substrate was loaded and the absorbance recorded in a time interval of thirty seconds (30 s) from time zero (Os) to three minutes (180 s) IA the following analysis | substrate | Bioca- Comment was made: talisador Experiment ID KG1112-021-20-CAR- | Benzoate rCAR Positive control 1SB sodium KG1112-021-20-CAR- | Pyramid 2PA rCAR acid KG1112-021-20-CAR- | Adipic rCAR 3AA acid KG1112-021-2 0-W- | Benzoate water Negative control using 1SB sodium water KG1112-021-37- Benzoate BL21 Negative control using BL21-1SB sodium host cells with empty vector KG1112-021-37- Acid BL21 Negative control using BL21-2PA pyelic host cells with empty vector KG1112-021-37- BL21 acid Negative control using adipic BL21-3AA host cells with empty vector

[00342] Results from the evaluation of the carboxylic acid reductase:

[00343] The results of the spectrophotometric evaluation measuring the consumption of NADPH in relation to time for each of the above evaluations is given in figure 13. When corrected for the previous rate of consumption of NADPH (negative control experiments using the substrate and the biocatalyst that kept the empty vector, table 2), it is clear that both adipic acid and pyelic acid are reduced at a higher rate than the model substrate, benzoate. Thus, it was successfully demonstrated that the enzyme carboxylic acid reductase has consumed NADHP and had activity in relation to sodium benzoate, adipic acid and pyelic acid.

Table 2 Consumption of NADPH | Previous NADPH consumption)

[00344] Thus, CAR obtained from Nocardia catalyzed the reduction of pyelic acid in pyelic acid semialdehyde with> 100% activity for benzoic acid. Other enzymes appropriate for the reduction of pyelic acid from EC 1.2.99.6 include, for example, that Streptomyces griséus (Suzuki et al., 2007, Grit and GriD is a carboxylic acid reductase involved in the biosynthesis of grixazone in Streptomyces griséus . "J. antibiot." (Tokyo), Jun; 60 (6): 380-7). In addition, ThnG, a non-acylating aldehyde dehydrogenase in Sphingomonas and Cupriavidus species, converts pyelic acid semialdehyde to pyelic acid during degradation and tetralin (See, figure 8) and can be reversible.

3.1.5 - Conversion of pimeloyl-CoA and / or pimeloyl- [acp] into pyelic acid by thioester hydrolases (EC 3.1.2.-), pimeloyl-Coa ligases (EC 6.2.1.-) or CoA-transferases, ( EC

2.8.3.-):

[00345] Enzymes that are able to catalyze the hydrolysis or transfer of CoASH or acyl [acp-vehicle-protein] thioesters include thioester hydrolases from EC

3.2.1.-, such as EC 3.1.2.2, EC 3.1.2.14, EC 3.1.2.18;

3.1.2.19, 3.1.2.20 EC 3.1.2.21, acid-thiol ligases of EC

6.2.1.-, such as EC 6.2.1.3, EC 6.2.1.5; EC 6.2.1.14, EC

6.2.1.20; EC 6.2.1.23 and CoA-transferases from EC 2.8.3.-, such as EC 2.8.3.12 and EC 2.8.3.13 or the ThnH gene product that catalyzes the reversible transfer of CoA in pyelic acid (Aroa López-Sánchez, Belén Floriano, Eloisa Andújar, Maria José Hernáez, and Eduardo Santero, 2010 - “Tetralin- Induced and ThnR-Regulated Aldehyde Dehydrogenase and BE- Oxidation Genes in Sphingomonas macrogolitabida Strain TFA. Appl. Environ Microbiol. 76: 110-1 18). Among these three families, thioesterases present the best candidates for the hydrolysis of pimeloyl-CoA due to the large number of “characterized” enzymes and their broad substrate specificity in terms of chain length (C-3 to C-26) and in terms of functional groups (Dicarboxylyl-CoA substrate).

3.1.5.1 - acid-thiol ligases (EC 6.2.1.-) and CoA-transferases (EC 2.8.3.-):

[00346] Acid-thiol ligases catalyze the formation of carbon-sulfur bonds with concomitant hydrolysis of adenosine triphosphate (ATP). CoA-ligases have not been tested vigorously or systematically for reversibility. No crystal structure is available, so the site-active structure is not known. Therefore, it is not clear whether AMP, pyrophosphate or magnesium ions would be needed for the reverse reaction, and in vitro analysis should be done in the presence and absence of combinations of these cofactors. Of course, the ultimate goal is to get the reverse reaction in every cell, and this can be more challenging. The last reactions on the route will, of course, take the balance out in the thiolitic direction. However, this can be a difficult reaction to reverse because the hydrolysis of ATP and the subsequent hydrolysis of pyrophosphate renders the reaction in the exergonic sequence. Therefore, it is important to measure the effect of different proportions ATP: AMP on the reversibility of the reaction in vitro before attempting to build the cells. If the ATP: AMP ratio is critical, it may be necessary to manipulate the cellular energy charge to make the intracellular ATP concentration low enough to allow the reverse reaction to continue. This, in turn, can cause additional difficulties with achieving good productivity, since it may be necessary to use ATP synthesis inhibitors or non-couplers to achieve low enough ATP concentrations. In principle, none of these problems are insurmountable, but the development of constructed biocatalysts can become complicated. The hydrolysis of acyl-CoA would therefore require production and ATP during the catalyzed acid-thiol ligase reaction which is thermodynamically unfavorable compared to the reverse reaction: o o o o don + AMP + PP, <> hn A, + CoASH + ATP

[00347] However, these enzymes are still useful if found to be reversible. Several CoA-ligases are known to act on pimeloyl-Coa:

v n v $? $? o o “o le í = gg e | | ú 2 3 = o o Z - “e E: E m E | Sm [86 At 5 o o a: H no 5: => Is 8 “| :: <<o o:: & o ': & la “ent ss Bs 8 lg lg ls 8 | & | "E? 2 E [2 | 4 à. 8 Eê8 Z sas EO So Sa and Á À On un - | É Z o. O.: Z“ o.

Z o. O. | & 2 is vs í o - * 7 o o o. 8 8 8 o Co o & 8 8 8 S <<S; 7 7 a | E | zios - 3 3 1 Í vB Ô ó <) º> o: & 8 <á SE 03 04 gos Sn 98 o <“SG o sESmmNidoe 33 158“ and TSE SERA AOS 5 fem is FaRBidabeeaeieasaks 2 fed o A adidas : aunts of É “o“ HT; N it of os: s; It is that of 8d No do Bão It is 2 to C of ms sl 3/9 lg 8le 2 ode is 8, | 8ê o F o E o: 1o7 | o B8E8E85o2 | 85 8 o A 2 Sa8TS SS BEE SE: ão S be Beige 2 goal 2 I love it E DU AO U ae TE and 2 ES | E 2: C; ; 4 Ú 2 le E 4º | E s 2 | "| E |: z E candle Slg | 8 z a Jon“ SE SS | S: BS —88 Sm SE 5 j E 4 ooo 7 cs In So and Es ole & ê USA E oo In els & 7 í: + qo SO So Ro F 3º gs 277 Ze Se ss í í:: Es E e Bla o 5 E 8 o as EX SE | I am ES ES 2 2 3 3 already ar à Ss 82 8 4 v 2 2 th [now be O + 2 88 |. TA SSH 8 ol 3rd 82a8s SEVEN Te Ro sm E magic If SgnoRo 2 foo, of7 dEaeREES 4 QE Lo Esmesie is not THOSE Sisadilido à 853/47 5 o 2 ERROR f «oio 4d Ao ". a Link o “Oppo 12 oo nm alSS 28 sR ES: 8 di2282d Bl El 2/2 4 SAS 2 to RR Ee 9 Ss É 5 ole] SEA SA RNA be nm 8 + Flális ese 28 NES Pg oLo ot salejsos 25 RE VPELXKXNOLAHAS ERAS: $ Se “28 Sl, ee 53 5 EM FS <P 38 À 3% lg: o. O. = Ss. :: S is to faith é | é & & & ú 3 $ s à | | - & és + & s QN 2 to 2

BioWw (Uniprot: P22822) - from Lysinibacillus sphaericus (Bacillus sphaericus):

[00348] Pyelic acid-CoA synthase (6-carboxyhexanoate-CoA ligase) was discovered in Bacillus sphaericus using cell-free extracts. She is involved in the biotin biosynthesis route. The enzyme activity was indirectly determined by a joint reaction with 7-keto-8-aminopelargonic acid synthase and microbiological analysis using Saccharomyces cerevisiae [1]. The gene encoding pyelic acid-CoA (bioW) synthase was identified along with 7-keto-8-aminopelargonic acid (BioF) synthase through complementary studies of E. coli mutants with a library carrying the genome sequences from B&B. sphaericus (Gloeckler R., Ohsawa T., Speck D., Ledoux C., Bernard S., Zinsius M, Villeval D., Kisou T., Kamogawa K, Lemoine 7Y. (1990), “Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin. "Gene - 87: 63-70]. BioW was overexpressed in E. coli and purified for homogeneity [Ploux O, Soularue P, Marquet A, Gloeckler R, Lemoine Y. ( 1992), "Investigation of the first step of biotin biosynthesis in Bacillus sphaericus; Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate." Biochem J. 287: 685-690]. The enzyme was active in the presence of pimelate disodium, ATP, COASH and MgCl; reaction products were pimeloyl-Coa and AMP, no ADP was detected. The enzyme was specific only for ATP and pimelate. Other “nucleotides (such as GTP, AMP) and alternative substrates (succinate , glutarate, adipate, suberate) were not accepted.

PauA (GenBank: AJ012480.1) from Pseudomonas mendocina 35 (EC

6.2.1.14):

[00349] The pimeloyl-Coa catabolic bond, pauA, is involved in the degradation of dicarboxylic acids (C6-Cl0) in Pseudomonas mendocina 35 [Binieda A., Fuhrmann M., Lehner BE. Rey-Berthod C., Frutiger-Hughes S, Hughes G, Shaw NM. (1999) "Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35". Biochem J. 340: 793-801]. The enzyme activates dicarboxylic acids which are subsequently metabolized via the beta-oxidation route. The enzyme was purified for homogeneity and the partial amino acid sequence was determined. PauA demonstrated a much wider range of substrates than bioW, and accepted pimelate (100% relative activity), adipate (72%) and azelate (18%). The enzyme was between one and two orders of amplitude more active than bioW involved in the biotin biosynthesis route. The pauA gene was identified and the ligase was successfully overexpressed in E. coli. Pseudomonas mendiocin pauA ligase was selected as an example from this class due to its substrate range and specific activity (preferred substrate is pyelic acid, Binieda et al., 1999. “Purification, characterization, DNA sequence and cloning of a pimeloyol -CoA synthetase from Pseudomonas mendocina 35 ". Biochem. J. 340: 793-801). o Ho o o no Ao + COA + ATP -> nor coa + AMP + diphosphate Cco2656 Cco106? Pimeloyl-Coa pyelic acid

[00350] An alternative may be the pimA of Rhodopseudomonas palustres, which was also expressed in E. coli and demonstrated to have ligase activity with a wide range of substrates (C7-Cl4 dicarboxylic acids), although pyelic acid is not a preferred substrate ( Harrison and Harwoo,

2005. The pimFABCDE operon of Rhodopseudomonas palustres mediates the degradation of dicarboxylic acid and participates in the degradation of anaerobic benzoate. Microbiol. 151: 727-736). For this reason the Pseudomonas mendocin pauA gene appears to be a better choice and was chosen for exemplification.

Sscs (Uniprot Q5JENT7) of thermococcus “Kkodakaraensis (EC

6.2.1.5):

[00351] The succinyl-CoA synthetase of Archaea Thermococcus kodakaraensis hypertermofilo (SCStx) is a heteromeric enzyme (0B.) Encoded by TKI1880 (a-subunit) and TKO0943 (B-subunit). SCS catalyzes the reversible conversion of succinyl-Coa to succinate and Coa concurrently with the level of ADP / GDP phosphorylation substrate. SCS: «k It is relatively specific, participating in relevant levels of activity for only a few acids. The activity levels and kinetic parameters of SCS: k, for succinate, isovalerate, and 3-methyl-thiopropionate suggest the involvement of this enzyme in the catabolism of Glu, Leu and Met. However, considering that ACS II “, also participates in high levels of activity for the last two substrates, the greatest physiological rule of SCS '., Is closer to the generation of ATP from succinyl-CoA. SCS :, It is a reversible enzyme and it showed 59% adipate activity of the native substrate (Shikata et al., 2007. J Biol Chem, 282: 26963-26970), therefore, it seems to be an appropriate candidate.

o o Ho + ATP + COÀ <DD> HO AA with Toostat o o cooos2 Succinyl CODON suceinila-Coa Pimeloil-CoA synthetase from Bacillus megaterium:

[00352] The enzyme has been purified about 34 times with 79% recovery [Izumi Y., Morita, H., Tani Y., Ogata K., (1974). Pimeloyl-Coa synthetase responsible for the first stage in the biotin biosynthesis by the microorganism. "Agr. Biol. Chem. ”, 38, 2257-2262]. The required reaction is pimelate, ATP, CoASH and MgCl ;. The enzyme was specific to pimelate only, and other dicarboxylic acids were not accepted. Interestingly, ATP could be replaced with ADP, suggesting that pyrophosphate cleavage is not essential. The amino acid and nucleotide sequences are not known. However, three complete genomes of Bacillus megaterium have been published since 1974. Therefore, a BLAST search for bioW counterparts has started. Unfortunately, this did not show any combination. Lavandula vera L plant pimeloyl-CoA synthesis:

[00353] The activity of pimeloyl-Coa synthase in lavender cell cultures has been described [6]. Although [* H] - pimelic acid is found as a precursor for biotin biosynthesis, there are no data available regarding the enzyme or the gene. Plant soluble acyl-Coa synthetase obtained from Psium sativum L .:

[00354] The activity was observed in the soluble fraction of pea protein extracts [7]. The enzyme required ATP / Mg ”* and CoASH. The molecular weight of the ligase was determined by the Western Blotting technique using polyclonal antibodies against Lysinibacillus sphaericus ligase. The enzyme accepted pyelic acid, as well as azelaic acid, but not nonanoic acid. GTP could be used instead of ATP, but no reaction was observed for ADP. The gene encoding soluble pimeloyl-CoA synthetase has not been identified.

[00355] Peroxisomal acyl-CoA synthesis of the plant, obtained from Psium sativum L .:

[00356] The enzyme was located in the peroxisomal protein fraction [GerblingH, AxiotisS, Douce R., (1994), “A new acyl-CoA synthetase, located in higher plant cytosol”, - JJ. Plant Physiol., 143: 561-564]. The pyelic acid was activated in pimeloyl-CoA and subsequently degraded into acetyl-Coa and malonyl-CoA via the beta-oxidation route. The substrate range of this enzyme has not been tested. A higher optimum pH was shown than that of soluble acyl-CoA synthetase from the same organism. Rattus norvegicus dicarboxylate-CoA ligase:

[00357] Dicarboxylic acids (C5-Cl6) were converted to their Coa esters by a dicarboxyl-CoA synthetase obtained from the microsomal protein fraction of a rat liver [Vamecg J., by Hoffmann E., Van Hoof F., (1985). “The mirosomal dicarboxyl-CoA synthetase” - Biochem. J., 230: 683-693]. The enzyme uses ATP, but not GTP. The reaction is inhibited by its products, and AMP is more inhibitory than pyrophosphate. The enzyme showed the highest activity for the C12 substrate. In some analyzes of dicarboxyl-Coa synthetase, the consumption plateau of CoA was followed by the release of CoA. This is an important observation because it suggests that the reaction may be reversible. However, this suggestion should be treated with some caution, as the protein sample may have been contaminated with CoA thioesterase. Although this enzyme can be promising, the BLAST search for the R. norvegicus genome sequence did not reveal any bioW or pauA homolog. An alternative in this search would be the need to identify the gene, for example, enzyme purification, N-terminal sequencing, primer design and PCR amplification.

Pimeloyl-Coa synthetase obtained from unidentified bacteria, strain LP-1:

[00358] The activity was observed in culture media of denitrifying bacteria from enriched must activated for growth in sodium nitrate and pimelate [Gallus C., Schink B., (1994), “Anaerobic degradation of pimelate by newly isolated denitrifying bacterial ”- Microbiology, 140: 409-416]. The enzyme was active in the presence of ATP and Mg ”. The reaction product was AMP in addition to ADP. The enzyme did not accept benzoic acid.

[00359] CoaA-transferases or less attractive candidates than thioesterases, due either to their limited substrate specificity or to a loss of gene / protein characterization so that for hydroxymethylglutarate succinate CoaA-transferase (EC 2.8.3.13 ), despite showing activity in relation to the C-6 derivative of pimeloyl-Coa, adipil-Coa (substrate specificity of a dicarboxyl-CoA acid transferase: dicarboxylic A obtained from rat liver mitochondria: Deana et al., “ Biochem. Int. ”, 1992, 26: 767-773). Only one succinyl-CoA: pimeloyl-Coa transferase has been reported, but the gene responsible for the activity is not available and the reported organism has not been identified

(Gallus and Schink (1994), “Microbiol.”, 10: 409-416 “Anaerobic degradation of pimelato by newly isolated denitrifying battery). Some enzymes obtained from this family can still be used, and the following enzymes have been identified as potential candidates: Coenzyme-A-Transferase (GCt) clutaconate obtained from Acidamicoccus fermentans (Uniprot Q59111):

[00360] Glutaconate coenzyme A-transferase (Gct) obtained from Acidamicoccus fermentans is an enzyme that catalyzes the formation of (R) -2-hydroxyglutaryl-Coa from acetyl-Coa and the free acid. Gct was characterized as a heterooctamer (a4B4) composed of two different subunits (GctA, 35725 Da and GctB, 29168 Da). Both the GctA, and GctB subunits were co-expressed in E. coli and showed to be functional as glutaconate CoA transferases (EC 2.8.3.13). Gct is also active on unsaturated and saturated substrates, as well as hydroxylated substrates (2-hydroxyglutarate), but has not been tested against C7 coenzyme substrates (Mack et al., 1994, “Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans ”). “Eur. J. Biochm. ”, 226: 41-51). This shows to be a promising target, since it was possible to alter the activity of the enzyme glutaconate CoA transferase from Acidamicoccus fermentans (GctA, EC2.8.3.12) (GLutaconate CoA-Transferase obtained from Acidamicoccus fermentans: Buckel et al., “Eur. J. Biochem.”, 1981; 118: 315-321) from a coA-transferase in a CoA-esterase by site-directed mutagenesis (Mack M., Buckel W. (1997). Conversion of CoA-glutaconate transferase from Acidamicoccus fermentans in an acyl-Coa hydrolase by site-directed mutagenesis (FEBS Let. 405: 209-212). This enzyme was successfully expressed in E. coli and used for enzymatic biosynthesis of glutaconyl-CoA, 3-methylglutaconyl-CoA, butynediol-CoA, 2-hyoxiadipioil-CoA, oxalocrotonil-CoA, and muconyl-CoA (Parthasarthy A., Pierik A., Kahnt 4. zelder O., Buckel W (2011), “Substrates specificity of 2-hydroxyglutyl-CoA dehydratase obtained from Clostridium synbiosum: in relation to production based on adipic acid bio,“ Biochemistry ”- 50: 3540- 35500). The crystal structure of GctAB was also pbulciated (Jacobte U., Mack M., Clausen T. Hubber R. Bauckel W. Messerschmidt A. (1977), Glutaconate CoA-transferase from Acidamicoccus fermentans: the crystal structure reveals homology with other CoA-transferase, “Structure” 5: 415-426 This transfer binds to the anions of the carboxy terminal via various bonds and hydrogen in serine residues, which can adjust to different chain sizes: oooooo HI6 ”Sicc04 * HITS o '? Ho "" oHs * HO Srs of conoz4 Ccozz14 conoas Co2411

[00361] Other appropriate CoaA-transferases include CoA-transferases from Rattus norvegious (Succinata-hydroxymethyl-glutarate CoA-transferase - EC 2.8.3.13), since it accepts adipyl-CoA as a substrate, with a higher affinity than than that observed for Oo succinyl-Coa, malonyl-CoA, glutaryl-CoA (substrate specificity of a dicarboxyl-CoA: dicarboxylic acid coenzyme A-transferase from rat mitochondria (Deana Biochem Int. ”, 1992, 26: 767 -773). Similarly, acetyl-CoA-: 5-hydroxypentanoate CoA-transferase obtained from Clostridium aminovalericum (EC

2.8.3.14) was not tested for activity in relation to C7 substrate, but did not show activity in relation to C3-C5 substrates and specific for saturated substrates (Eikmanns and Buckle, 1990, “Properties of 5-hydroxyvalerate CoA-transferase from Clostridum aminovalericum ", - Biol. Chem., 371: 1077-1080). The genes responsible for this activity have been listed.

o o x o Hi67S-200A: took S> HOMCCHIZ * nom A s-cos conc24 CoZ804 Ccon033 co3s237

[00362] Exemplification of reversible hydrolysis of pimeloyl-CoA in pyelic acid by acid-thiol ligases and Co-transferases:

[00363] The enzymes below were selected from the list above, are selected as examples: Source organism | Synthesis of Protein Name UniProt gene = Reference accession code Thermococcus | BDIGENEO0O041 Large subunit of Q5JENT kodakaraensis succinila-CoA synthase EC 6.2.1.5 BDIGENEO0042 Beta subunit of Q5JIA9 acetyl-CoA synthetase II Acidaminococcus | BDIGENE0043 Subunit of 059111 fermenters Glutaconate CoA-Ec 2.8.3.12 transferase A (GctA) BDIGENEO0044 Subunit of Q59112 Glutaconate CoA-transferase B (GctB) Pseudomonas BDIGENEOO045 | Pimeloyl-CoA synthase Q9ZER2 mendocin EC (pauA)

6.2.1.14

[00364] The genes were optimized at the codon for E. coli and expressed in BL21 (DE3) as previously reported (Binieda et al., (1999) “Biochem. J.”, 240: 793-801; Shikata et al., (2007) A new ADP-forming succinyl-CoA synthetase in Thermococcus kadakaraensis structurally related to the nucleoside - archaeal acetyl-CoA diphosphate-forming synthetase. “J. Biol. Chem.”, 282: 26963-26970). His-target protein was purified on 1 ml of Histrap columns. The enzyme obtained from Pseudomonas mendocina was completely insoluble and only trace amounts could be doubled, so only the enzymes obtained from Acidaminococcus & E Thermococcus were used to evaluate the activity with its native substrates. The activity for both enzymes was confirmed using colorimetric evaluation monitoring the formation of CoA esters from their natural substrates, (E) - glutaconate and succinate, by measuring absorbance at 520 nm and 405 nm, respectively.

[00365] Biotransformation reactions to assess the disappearance of Adipoyl-CoA and pimeloyl-coa and formation and adipic acid or pimelic acid were carried out in 1 ml reactions by adding 30ul of the protein solution to 300 ml of the reaction mixture which contained (per ml) in the reaction mixtures, as described below: [CC GFroteina - [13/11 [01/02] 05 [03/00] 01/02] 05] Add stock volume AO AD AM PM to achieve : 20 Mm sodium phosphate, 16 16 16 16 16 16 pH 7.0 Im adipoilecoa - [16 Iwo De [=] mM AMP Le Te Tere Tee) [EmA Ce Te E Ee) 5 mM sodium acetate | 4 | - | - [4 | - | - | 2.5 mM MgCl; (Added 2 2 2 2 2 2 last)

[00366] The negative controls contained 30 ul of the elution buffer in place of the enzyme solution. The reaction mixtures were incubated at 70ºC (41/42) or room temperature (43/44; 45) for 5 hours, diluted with 570 µl of water and evaluated by LC-MS.

[00367] All 3 enzymes could hydrolyze pimeloyl-CoA in pyelic acid (See, figure 26). Despite the fact that only the trace amounts of the soluble enzyme of Pauà could be produced, this enzyme forming pyelic acid from pimeloil-CoA. This confirmed that the EC 6.2.1.14 acid-thiol ligases are reversible in reverse, and can be used to prepare pyelic acid from pimeloyl-Coa. In addition, CoA transferases can be used to produce pyelic acid from pimeloylcoa.

3.1.5.3 - Thioester Hydrolases (EC 3.1.2 .-) .-)

3.1.4.3.1 - Selection of appropriate enzymes:

[00368] Thioestrases are hydrolytic enzymes that catalyze the hydrolysis of Coa or [acp] -thioesters: o o o o its 00 ho -> PN Ao + CoASH

[00369] Thioesterases are ubiquitous in all organisms acting on CoA oul [acp] activating carboxylic acids and belonging to EC 3.1.2, including EC 3.1.2.2; EC 3.1.2.28; EC 3.1.2.19; EC 3.1.2.20 for ester-CoA hydrolases, although [acp] -thioester “hydrolases are observed in EC 3.1.2.14 and EC 3.1.2.21. Only two thioesterases (ACOT8 and ACOTA4) were tested for activity with respect to dicarboxylyl mono-CoA esters such as succinyl-CoA.

EC Enzyme Source Substrates Purification Size (kDa) ACOT8 3.1.2.27 Mus Wide range, CoA 50 Yes, his- fusion | (PTE-2) Imusculus | straight chain, tag Coa bile acid homogeneity | and ACOT5 branched chain acyl-CoA 3.1.2.2 Mus Acil-CoA 46.6 Yes, his- fusion | (PTE-Ic) | Hidrolase | musculusl medium chain, tag palmitoil-CoA | lhomogeneity ACOT3 3.1.2.2 Mus Acil-CoAs of 47.4 Yes, his fusion (PTE-Ta) | Imusculusl hydrolase long chains tag palmitoul-CoA lhomogeneity | ACOT4 3.1.2.3 Mus Succinila-coA e | 36 (active | lYes, fusion his- | (PTE-Ib) | Thioesterase Imusculus | glutaryl-CoA as tag succcinyl-CoA only dimer) lhomogeneicidad Thioesterases acyl-CoA 8 peroxismal ACOT8 (formally PTE- 2) from Mus musculus:

[00370] ACOT8 is a homologue of human PET1l thioesterase. The enzyme was overexpressed as a His-Tag fusion protein in E. coli and purified for homogeneity using His-Tag column chromatography [Westin MA, Hunt MC, Alexson SE. (2005) The identification of a succinyl-CoA thioesterase suggests a novel pathway for succinate production in peroxisomes. J Biol Chem. 280: 38125-38132]. The activity was inhibited at substrate concentrations> 5-10 UM for long chain acyl-CoA, but the addition of BSA prevented inhibition. The thioesterase substrate range was tested using saturated and unsaturated linear chain acyl-CoAs (C2-C20). The highest activity was for C10-C118, bile acids (thihydroxyprostanoil-CoA, coloyl-CoA and chenodeoxicoloil-CoA and acyl-CoAs of branched chains). Km values for many of the tested substrates were very low, strongly suggesting that ACOT8 had a very broad substrate specificity and can accept many of them. ACOT8 was strongly inhibited by CoASH with an IC50 of 10-15 UM. ACOT8 was also tested for activity in relation to dicarboxylyl mono-CoA derivatives and, preferably, hydrolyzing long chain substrates (activity increased with C3 chain length from C3 to C12) [Hunt MC, Solaas K, Kase BF, Alexson SE. (2002) Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism. J Biol Chem. 277: 1128-1138]. Mus musculus ACOT3 (Formally PTE her):

[00371] ACOT3 was expressed in E. coli and purified for homogeneity [19]. The enzyme demonstrated activity for C8 to C26 monocarboxylic acid esters, with the highest activity in relation to long chain substrates (C1l6). The enzyme did not accept the bile acid esters. There was no inhibition with free CoASH. Dicarboxylic acid esters have not been tested. Mus musculus ACOT5 (Formally PTE-lc):

[00372] ACOT5 was expressed in E. coli and purified for homogeneity [Westin MA, Alexson SE, Hunt MC. (2004 Molecular cloning and characterization of two mouse peroxisome proliferator-activated alpha receptor (PPARalpha) - regulated peroxisomal acyl-CoA thioesterases. J Biol Chem. 279: 21841-21848]). The enzyme demonstrated activity for C6 to C20 monocarboxylic acid esters, with the highest activity in reaction to medium chain substrates (C10). The enzyme did not accept the bile acid esters. There was no inhibition with free CoASH. Dicarboxylic acid esters have not been tested. ACOT4 peroxisomal acyl-CoA thioesterase 4 (Formally PTE-1b) from Mus musculus:

[00373] ACOT4 was expressed as an expression in E. coli and purified for homogeneity [18]. The enzyme demonstrated a limited substrate range, accepting succinyl-Coa and glutaryl-Coa. The reaction was not inhibited by CoASH at concentrations up to 500 UM.

[00374] ACOT8, ACOT5 and ACOT3 were then excellent candidates for the hydrolysis of dicarboxylyl-CoAs and other acyl-CoAs that are involved in the pyelic acid biosynthesis route.

3.1.5.3.2 - Cloning, expression and biotransformations with selected thioesterases:

[00375] Mus musculus Acot8 thioesterases (GeneBank NM 133240 WT mRNA sequence; 65-1027 fragmentomRNA) and Haemophilus influenzae YciA (WT gene sequence (GeneBank L42023, fragment 878596-879060, EC 3.1.2.20) were selected as example for the conversion of pimeloyl-CoA to pyelic acid, due to its specificity in relation to dicarboxylyl-CoA substrates (The identification of succinyl-CoA thioesterases suggests a new route for the production of succinate in peroxisome: Westin J. et al. Biol. Chem 2005, 280, 38125- 38132; Divergence of function in the hot dog fold enzyme superfamily - the bacterial thioesterase YciA: Zhuang et al. Biochemistry 2008, 47, 2789-2796) Both genes have been identified from their respective genomes hosts and cloned and successfully expressed in Escherichia coli (Characterization of an acyl-CoA thioesterase that functions as a major regulator of peroxisomal lipid metabolism: Hunt et al. J. Biol. Chem. 277, 1128-1 138; Divergence of fu nction in the hot dog fold enzyme superfamily - the bacterial thioesterase YciA: Zhuang et al. Biochemistry 2008, 47, 2789-2796).

[00376] Both genes were selected as allele-wild sequences for the construction of the corresponding expression vectors in E. coli and used the transformation of E. coli BL21 (DE3). The expression of thioesterases Haemophilus influenzae YciA was achieved in E. coli (figure 14h) but not of the dioesterase Acot8 of Mus musculus. The protein band in the expected size (17 kDa) was clearly detected after IPTG induction in the soluble fraction (Columns 2 to 3) and the insoluble fraction (Columns 6 and 7). No corresponding protein was observed in the experiment with the negative control (Columns 4 and 8) suggesting that the thioesterase protein of expected size of 17 KkDA from Haemophilus influenzae YciAÃ was successfully expressed in E. coli from the produced I8 construct. The soluble expression of the YciA protein appears to increase over time between 4 hours and 24 hours after the induction incubation (Columns 2 and 3) while the insoluble expression appears to decrease over the same period (Columns 6 and 7). Therefore, the 24-hour soluble sample proved to be a better candidate for monitoring activity of Haemophilus influenza YciA thioesterase.

3.1.5.3.3 - Synthesis of pimeloyl-CoA:

[00377] The sodium salt of coenzyme A (150 mg, 195 umol) was suspended in a mixture of acetone (22, 5 ml) and water (225 UL). The suspension was cooled to 0 ° C in gel and adjusted to pH 8 with 200 mM sodium bicarbonate solution. Hydrochloric acid (586 umol, 3 equiv.) Was added slowly to the suspension with stirring while maintaining the pH of the reaction at 8, by complementary addition of the sodium bicarbonate solution. The reaction mixture was allowed to stir at 0 ° C for an additional 1 hour. Once the coenzyme a has been consumed, as determined by monitoring the HPLC reaction, the reaction mixture was concentrated in vacuo, and the white residue was resuspended in water (20 mL), adjusted to a pH of 3 with 5M of phosphoric acid and extracted with diethyl ether (3 x 20 mL). The remaining aqueous layer was then purified by solid phase extraction (SPE) using a polymeric reverse phase with the 19/20 mL Strata x 33 um capsule (cartridge) provided by Phenomenex.

[00378] The SPE capsule was prepared with methanol and equilibrated with 10mMM of sodium phosphate buffer (pH 4.2) before use. The aqueous layer containing the crude acyl-CoA product was loaded into the capsule. Washings with 10 mM sodium phosphate buffer (pH 4.2) (3 x 5 ml) were performed, followed by a gradual gradient using 5, 10, and 20% MeOH / buffer elutions (3 x 5 ml). Each fraction was collected and analyzed by HPLC. Fractions containing the product were only combined and concentrated in vacuo. The isolated products were used directly in biotransformation without further purification. 'H NMR or LCMS were used to characterize the products.

3.1.5.3.4 - Biotransformation of pimeloyl-CoA into pyelic acid:

[00379] Biotransformations were performed in a final volume of 2 mL. Biotransformations comprising 1 mM substrate, 200 mM KCl and 50 mM potassium Hepes (pH 7.5). The negative controls comprising the cells housing the empty vector. The biocatalyst was loaded as a cell-free extract to result in a final concentration of -159 / L in the entire cell equivalent. The reactions were carried out at room temperature (20ºC) in tubes

15 mL falcon with stirring. Each biotransformation was sampled after 1 hour, 4 hours and 24 hours and analyzed by HPLC (Agilent 1100) on a 250 x 4.6 mm, 5 micron Phenomenex Luna C18- column. Before injection, each sample was heated at 100ºC for 5 minutes to stop the reaction, centrifuged at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter. The filtrates were analyzed by HPLC at 260 nm to disappear pimeloyl-CoA (Rt 12.39 minutes) and the formation of Coa (Rt 7.98 minutes), both of which absorb, strongly in this wavelength, while the pyelic acid does not was UV detectable at 260 nm. The formation of pyelic acid was confirmed by LC-MS.

[00380] A low level of antecedent hydrolysis was observed in the negative control, which can be attributed to the low levels of endogenous thioesterase activity in E. coli hosting the empty vector. Complete hydrolysis of pimeloyl-coa in pyelic acid and acetyl-Coa was observed after 1 hour of reaction.

3.1.6 - conversion of semialdehyde from pyelic acid to 7-aminoheptanoic acid by O-transaminases (EC 2.6.1.-) and conversion of a-ketosuberate to a-aminosuberate by amino acid aminotransferases (EC 2.6.1.-) :

[00381] Transminases or aminotransferases are enzymes that catalyze the reaction between an amino acid and a keto acid. This reaction involves removing the amino group from the amino donor, leaving it through an a-keto acid, and transferring it to the a-keto acid receptor and converting it to an amino acid. They are important enzymes in the synthesis of amino acids and are ubiquitous throughout nature. Many of these enzymes contain pyridoxal phosphate (PLP) as a cofactor. These enzymes belong to a large diverse family covering a wide range of cellular functions, using a wide range of substrates.

3.1.6.1 - Selection of transaminases:

[00382] A number of candidates, identified by the literature search, BLAST search and protein database are appropriate enzymes for the conversion of pyelic acid semialdehyde to 7-aminoheptanoic acid. Source Number Name Gene Protein Enzyme / No. access available | purified

EC EC 2.6.1.19/YP 001823341.1 | Streptomyces Yes Yes gray, LysN Q72LL6 Thermus thermophillus) Yes Yes AADaT Q64602 Rattus Yes Yes norvegicus EC 2.6.1.19 Gray Streptomyces 4-aminobutyrate transaminase:

[00383] This enzyme was first studied in 1985 and was observed to catalyze the reaction: 4-aminobutanoate + 2-oxoglutarate - 4-oxobutanoatot + tglutamate The enzyme has a relatively broad range of substrate, including 6-aminohexanoate and 7-aminoheptanoate, using 2-oxoglutarate as the amino receptor, and formation of the corresponding semialdehydes (Yonaha, K., K. Suzuki, and S. Toyama, 4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties. Eur JJ Biochem, 1985 146 (1): p. 101-6). Although this is very promising, the reversibility of these reactions has not been tested. Therefore, the reverse reactions are tested in vitro, using glutamate as the amino donor, since this would be provided with 7-oxoheptanoate 7-aminoheptanoic acid. The enzyme was purified on S. griséus IFO 3102 (S. griséus subspecies griséus) and was found to have 100 KkDA, consisting of two identical subunits of approximately 50 kDa each. The cofactor is pyridoxal 5 '"- phosphate. A protein is deposited in the Pubmed database (Table 1). The gene encoding it (SGR 1829), annotated as a putative 4-aminotransferase, present in the S. genome sequence. gray IFO 13350, which is closely related to IFO3102. Although not clear, it is highly equal that this protein is a homologue close to the enzyme described and studied (Yonaha, K., K. Suzuki, and S. Toyama, 4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties. Eur JJ Biochem, 1985. 146 (1): p. 101 -6). In view of the available data, the substrate range, this protein is a very good candidate for testing as an appropriate aminotransferase enzyme EC 2.6.1.39 Thermus thermophilus aminoadipate transaminase (972LL6):

[00384] This is an enzyme with homology to alpha-aminoadipate aminotransferase (AAAAT) from mammals and is taught to be involved in the synthesis of lysine in the bacterium Thermus thermophilus HB27 (2,3]. Kinetic data are available for such substrates with 2-oxoadipate, 2-oxoisocaproate, alpha-aminoadipate and even aromatic amino acids [3] .The protein was expressed and purified in E. coli and was found to exist in vitro mainly as a homo-dimer, with each sub-unit of -44 kDa in size (Miyazaki, T., et al., alpha-Aminoadipate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus. Microbiology,

2004. 150 (Pt 7): p. 2327-34). The broad substrate specificity, its prokaryotic origin and the availability of the coding gene suggest that it may be a good candidate for testing with l. It is a very well studied enzyme and its crystal structure is available (PDB ID: 2Z2P7), which offers a mechanical view within the substrate specificity (Ouchi, T., et al., Dual roles of a conserved pair, Arg23 and Ser20 , in recognition of multiple substrates in alpha-aminoadipate aminotransferase from Thermus thermophilus. Biochem Biophys Res Commun, 2009. 388 (1): p. 21 —-7; Tomita, T., et al., Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus, Proteins, 2009. 75 (2): p. 348-59). Aminotransferase aminoadipate from Rattus norvegicus (Q64602): This enzyme is similar to the enzyme from Thermus thermopphilus previously described. It is noted as a quinureine / aminoadipate aminotransferase, although there is some evidence to suggest that two distinct proteins carrying out these reactions (Mawal, MR and DR Deshmukh, Alpha-aminoadipate and kynurenine aminotransferase activities from rat kidney. Evidence for separate identity. J Biol Chem ,

1991. 266 (4): p. 2573-5). The protein was purified from rat kidney and expressed in human enbryonic kidney fibroblast cells was observed to be around 100 kD in size. The enzyme can accept 2-oxoadipate as a substrate. Previous studies (presumably on the same enzyme) indicate that it is capable of using DL-2-aminopymelic acid as an amino donor, with 2-oxoglutarate as a receptor in a reaction that is apparently reversible (Deshmukh, D.R. and

S.M. Mungre, Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney. Biochem J, 1989. 261 (3): p. 761 -8). The same paper indicated that pyelic acid can be an activity inhibitor with a 30% reduction in activity in the presence of pyelic acid.

[00385] The above three enzymes could be suitable for final transamination to result in 7-aminoheptanoic acid. The enzyme of S. griséus is a good candidate and the process of cloning, expression and purification would not be particularly difficult. The same is true for T. thermophilus LysN, which appears to have a wide range of substrates. The Rattus norvegicus enzyme would be more stimulating, but the activity data suggests that this enzyme may be able to use the specific substrates required. Transaminase 5-aminovalerate (EC 2.6.1.48):

The diamino aminotransferases of EC 2.6.1.48, as well as the 5-aminovalerate transaminase are also suitable catalysts for the preparation of 7-aminoheptanoic acid from pyelic acid semialdehyde.

3.1.62 - Expression of selected aminotransferases:

[00387] Two genes have been selected for the conversion of both pyelic acid semialdehyde to 7-aminoheptanoic acid, and the conversion of suberate a-keto to a-amino suberate: (1) IlvE-Omega Vf fusion (SEQ ID 1 and 2) and (2) IlvE-Ad fusion optimized omega SEQ ID 3 and 4). These genes were prepared in plasmids that incorporate the fusion of omega-aminotransferase with the 5'- end of the branched chain IlvE amino transferase, which previously showed high level of expression in E. coli (See, figure 23 and figure 24). It has been shown that the introduction of the IlvE fragment significantly increases the expression of aminotransferase genes. Another heat induced plasmid containing a branched-chain aminotransferase gene with the IlvE fragment was also tested (ING66) in the activity evaluation.

[00388] Five additional genes (Bacillus weihenstephanensis KBAB4, Pseudomonas aeruginosa (gi9951072), Bacillus subtilis (gil6078032), Pseudomonas syringae class III, Rhodobacter sphaeroides class III) were selected (DSM patent (Preparation of 6-aminocaproic acidic 5 acid: DSM - US patent 2011-0171699 Al) These enzymes have shown some activity in the conversion of a-keto pyelic acid to amainoic pyelic acid and may also be active in the conversion of pyelic acid semialdehyde to 7-amino heptanoic acid (See , SEQ ID 5-14).

[00389] SDS-PAGE analysis of IlvE-Omega Vf fusion (pING2022) (Figure 23) and optimized fusion of IlvE-Ad Omega (pING2030) (Figure 24) of the expression proteins in E. coli BL21 as before. A light band of expected size is seen in both IlvE-Omega Vs fusion proteins (pING2022) (Figure 15, Columns 5 and 6) and Omega optimized IlvE-Ad fusion (pING2030) (Figure 15, Columns 8 and 9). There is no corresponding band in the negative control, confirming the expression of the IlvE-Omega Vf fusion protein and the omega-optimized IlvE-Ad fusion protein. Evaluation of the activity of the IlvE-Omega VÍf fusion protein (PpING2022) and the optimized IlvE-Ad fusion protein (pING2030) aminotransferase:

[00390] Recombinant Vf aminotransferase was used to evaluate for activity on T7-aminoheptanoic acid. Three transformations were prepared by examining the effect of enzyme loading on the conversion of T7-aminoheptanoic acid and sodium pyruvate to L-alanine. The detection of L-alanine in biotransformation would provide positive evidence for the desired specificity of the aminotransferase in 7-aminoheptanoic acid. The reaction scheme representing the conversion of 7-aminoheptanoic acid aminotransferase into pyelic acid semialdehyde represented below: eos echos Pyruvic acid Transaminase Lralanine FO ——————————> EF —DA2 / Y- + +

H AANR ADS COH ANA color H, N o 7-aminoheptanoic acid Semialdehyde of pyelic acid

[00391] Three biotransformations (3ml volume) containing SU mM of sodium pyruvate and 10mM of 7-aminoheptanoic acid. The reactions were kept in an orbital incubator at 30ºC and aliquots removed periodically from the biotransformations for analysis. Acid Pyruvate ID 7- Sample load sodium / mM aminoheptanoic / mM | enzyme v / v% Re0212-40-1 Roozi2-402 | - 50 | 10 f / 66 ReO212-40-3

[00392] The aliquots (1ml) were removed from the biotransformations and heated at 95ºC for 5 minutes to precipitate the soluble protein. The sample was subsequently centrifuged and the supernatant filtered through a 0.2 micron filter before HPLC analysis. The HPLC analysis used a pre-column derivative method based on the formation of the chemical adduct with beta-mercaptoethanol and orthophthaldehyde. The external standards of L-alanine and 7-aminoheptanoic acid were used to determine the concentration of 7-aminoheptanoic acid and L-alanine in biotransformation.

[00393] The results (Figure 16) clearly show the formation of L-alanine in biotransformation providing convincing evidence for aminotransferase activity and specificity with Vf aminotransferase. The concentration of 7-aminoheptanoic acid decreases with time that is consistent with the expected aminotransferase activity. The rate of 7-aminoheptanoic acid increases with increased concentration of the enzyme which is again consistent with the expected transaminase activity. The amount of L-alanine formed is not combined by the 7-aminoheptanoic consumed, this could be caused by the alternative biochemical degradation of L-alanine formed by other non-aminotransferase enzymes present in the E. coli extract.

[00394] An additional examination of the amino transferases was performed to evaluate the activity in relation to 2-amino suberate.

À co H, N co, H Pyruvic acid Transaminase L-alanine + Fr + COo, H Co, H À A IN color ANA or H, N Suberic L-2-amino acid Subteric 2-keto acid

[00395] A conversion of L-2-aminosuberic acid aminotransferase to 2-ketosuberic acid provides an opportunity for modulated biotransformation.

[00396] Six biotransformations were performed by examining pING 2022, PpING2030 and ING66 (Ilve branched chain aminotransferase, EC 2.6.1.42, already built in the strain collection). Each biotransformation contained 10mM of submeric DL-2 amino acid, 50 mM of sodium pyruvate and high and low loads of free cell extract (CFE) as indicated below, N.B. ING 66 used as total cells:

AND

[00397] The six biotransformations (3ml volume) were kept in 30ºC in an orbital incubator and aliquots removed periodically from the biotransformations for analysis. The aliquots (1 ml) were removed from the biotransformation and heated at 95ºC for 5 minutes to precipitate the soluble protein. The sample was subsequently centrifuged and the supernatant filtered through a 0.2 micron filter before HPLC analysis. The HPLC analysis used a pre-column derivation method based on the formation of the chemical adduct with Boc-cysteine and orthophthaldehyde. The external standards of DL-2-amino submeric acid and L-alanine were used to determine the retention time of enantiomers of 2-amino submeric acid.

[00398] The results indicate a reduction in the concentration of submerged L-2-amino acid and an enantio enrichment in submerged D-2-amino acid through all six biotransformations. This effect is consistent with the action of the aminotransferase on the L-enantiomer, since the aminotransferase tested here are known to exhibit L-stere selectivity. No measurement of L-alanine was observed by HPLC that would result from pyruvate transamination, however it is likely that the L-alanine formed was further degraded by another host protein present in biotransformation as previously observed in the 7-aminoheptanoic acid reactions .

[00399] Biotransformations ING66 (rc02120461 and -2) showed significantly higher levels of degradation of L-2-aminosuberic acid compared to the pING2022 and pING2030 aminotransferases. The ING66 branched chain amino acid aminotransferase is already known to have excellent selectivity for L-2-aminoglutarate (L-glutamic acid) which is a short (C5) homologue of submerged L-2-amino acid, so this preference in activity for ING 66 is not expected. Production of 7-aminoheptanoic acid from pyelic acid semialdehyde:

[00400] Based on the observed activity of Vibrio fluvialis M-aminotransferase activity (HQO418483.1 accession number (WT gene sequence: fragment 325-1686)) on 7-aminoheptanoic acid, the reaction was carried out in the opposite direction, that is, conversion of semialdehyde from pyelic acid to 7-aminoheptanoic acid. Both Vibrio fluvialis M-aminotransferase enzymes, as well as the purified N-terminal-His-Tag from Bacillus wihenstephanensis were evaluated. The acid The amino transaminase from Bacillus weihenstephanensis (accession number JAl14119.1) was expressed and purified using standard techniques. The reactions were carried out with both adipic acid semialdehyde and pyelic acid semialdehyde enzymes and product formation (6-aminocaproic acid and 7T-aminoheptanoic acid) were confirmed by HPLC (Agilent 1200, GraceSmart RP C18 column). General procedure for synthesis of adipic acid semialdehyde and pyelic acid semialdehyde:

[00401] The synthesis of semialdehyde C6 & C7 was carried out according to the general reaction scheme below, based on the published methods (Synthetic communications, 21 (8 & 9), 1075-1081 (1991): oo GH, SO, To o ny o MeoH remo PCC ta n OoMe poem O n OMe 3a n = 1 methyl-6- o = tyl-6- K2S2O; o 1 methyl 6 oxyhexanoate idroxyhexanoate: DP 2b n = 2 methyl-7- 4a n = 2 methyl- J- H, SO, oxoheptanoate 2 to hydroxyheptanoate MeOH 7 Ligase, pH8 1 H or AI Ahora 4a n = 1 6-oxyhexanoate (Na salt) 4b n = 2 7-oxoheptanoate (Na salt) Preparation Methyl-6-hydroxyhexanoate ( 2a):

[00402] Eercaprolactone la (509, 0.425 mol, 1 equiv.) Was dissolved in methanol (500 ml). Concentrated sulfuric acid (1mL) was charged and the solution was heated and the solution was heated to reflux overnight. Complete conversion to 2a was determined by GC-MS. The reaction was allowed to cool to RT and water (700 mL) was charged. The product was extracted into diethyl ether (3 x 200 mLO, dried over magnesium sulfate and concentrated to provide a clear, colored liquid (40.7 g). The crude product was distilled under high vacuum (b.p. =

120ºC, 7 mbar) to provide a 2 to (32, 9g) colored oil that was used directly in the next stage. Preparation of Methyl-7hydroxyheptanoate (2b):

[00403] Concentrated sulfuric acid (1mL), water (120 ml) and methanol were cooled to 15ºC on ice. Potassium persulfate (365.6 9, 1.34 mol, 3 equiv.) Was added slowly with stirring. Cycloheptanoate 1b (50 dg, 0.447mmol, 1 equiv.) In methanol (150 ml) was added dropwise over 45 minutes. The reaction was left to warm RT overnight with stirring. Complete conversion to 2b was determined by GC-MS. At this point, the work product described in 2a was followed. General procedure for PCC oxidation of hydroxy esters 2a / b to prepare ester aldehydes 3a / b:

[00404] Pyridinium chlorochromate (PCC) (60 g) and silica gel (60 g) were loaded into a 1L RB flask along with dichloromethane (200 mL). The suspension was concentrated to dryness to obtain a fine powder. Dichloromethane (500 mL) was loaded along with a 3À molecular sieve (70 g). The suspension was cooled to 0 ° C on ice.

[00405] Each hydroxy ester (2a, 32.69, 2b, 379) was dissolved in dichloromethane (30mL) and added dropwise in a PCC / silica suspension over 30 minutes. After addition with stirring for 30 minutes, complete conversion to Aldehyde ester 3a / b was observed by GC-MS. The crude reaction mixture was filtered through a pad of celite. The washes with diethyl ether (5 x 200 mL) were done to ensure the entire product was washed in a light, dark brown filtrate. The filtrate was then passed through a silica pad to remove colored impurities. Additional washes of diethyl ether (e x 100 ml) from the silica pad were done. The combined filtrates were concentrated in vacuo to provide a pale yellow-green liquid (3 a, 24.5 g, 3 7.99). The crude product was purified by short high vacuum distillation routes (3a bp = 98ºC, 8 mbar, 3b bp = 106ºC, 7 mbar) to provide a light colored distillate (3 a, 16.9 g, 3b, 49 gq ). The products were characterized by 'H and “C NMR analysis.

General procedure for hydrolysis of 3 a / b to make C6 / C7 4a / b acid semialdehyde using Candida Antarctica immobilized lipase acrylic resin:

[00406] Each aldehyde ester 3a / b (19, 6.9 mmol, 1 equiv.) Was suspended in 50 mM sodium phosphate buffer (pH 8) (20 ml). The immobilized lipase resin (100 mg) was loaded and the suspension was heated to 50 ° C and stirred for 45 minutes. After this time, GC-MS indicated that the aldehyde ester of the starting material 3a / b was consumed. The suspension was cooled to RT, the lipase beads were filtered and the resulting filtrate was concentrated in vacuo to provide the C6 / C7 4a / 4b semialdehyde in phosphate buffer. The products were characterized by H NMR.

General procedure for 3a / b hydrolysis to make C6 / C7 4a / b acid semialdehydes using Antarctic Candida immobilized lipase acrylic resin:

[00407] Each aldehyde ester 3 a / b (19, 6.9 mmol, 1 equiv.) Was suspended in 50 mM sodium phosphate buffer (pH 8) (20mML). The immobilized lipase resin (100 mg) was loaded and the suspension was heated to 50ºC and stirred for 45 minutes. After this period, GC-MS indicated the aldehyde ester starting material 3 a / b was consumed. The suspension was cooled in RT, the lipase beads were filtered and the resulting filtrate was concentrated in vacuo to provide the crude semialdehyde C6 / C7 4a / 4b in phosphate buffer. The products were characterized by 'H NMR. Biotransformation conditions of V.f. acid transaminase WO-amino Laboratory book reference Kg 1112-021-10 Substrate concentration 5 mM (6-0x0 hexenoic acid) mM (7-oxo heptanoic acid) L-monosodium glutamate (100 mM amino donor) Biocatalyst load (9 / L) with | 15 g / L base equivalents in every cell equivalent throughout the cell * (WCE) (free cell extract) Temperature (grade O) Final reaction volume * The term whole cell equivalents (WCE) is the term that is used for estimate the amount of biocatalyst present in the cell-free extract for cell lysis. This is calculated by resuspending a known weight of all wet cells in a defined volume of lysis buffer. For example, if 1 g of wet total cell pellets is suspended in 1 ml of lysis buffer, the WCE concentration is 1009 / L. typically, we prepare a stock suspension of biocatalysts on this path and dilute this stock when necessary to obtain the desired biocatalyst concentration in each biotransformation. Protocols for in situ preparation of C6 / C7 semialdehyde substrates used in biotransformation:

[00408] 10mM of C6 and C7 semialdehyde stock solutions were prepared before testing. The following protocol was used to prepare these stock solutions: 10 mM of methyl ester aldehyde was suspended in 50 mM of sodium phosphate (pH 7) (50 mL). The acrylic lipase resin immobilized from Candida antarctica 910 mg) was loaded. The suspension was heated to 50ºC and stirred for 1 hour. The TCL analysis indicated the complete consumption of the starting material after this period. The reaction was cooled in RT, the beads were filtered and the filtrate, containing -10mMM of semialdehyde, was used directly in each biotransformation. V.f. acid transaminase biotransformation protocol. W-amino:

[00409] 6 biotransformations were then performed, each with a final volume of 5 ml. Each biotransformation comprised 5mM of semialdehyde, 100 mM of L-monosodium glutamate and the biocatalyst, which was loaded as a cell-free extract to result in a concentration of 15 g / l of equivalents from the entire cell. The reactions were carried out at 30ºC in 15 mL Falcon tubes with stirring at pH 7. Each biotransformation was periodically sampled and analyzed by HPLC. Before injection, each sample was heated to 100ºC for 5 minutes, stirred at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter. List of biotransformations carried out for V.f. acid transaminase. W-amino:

Biocatalyst Substrate ID Comment experiment KG1112-021-10- | 6-0xo- acid | Free extract of 86 hexanoic cell transaminase of Mw-amino V acid. KG1112-021-10- 6-O0xo- acid | E.coli BW25113 Negative 96 Adadax hexanoic control using host cells with empty vector KG1012-021-80-6 6-Oxo- acid Hexanoic water KG1112-021-10- | 7-Oxo- acid | Free extract of 87 heptanoic cell transaminase Mw-amino V acid. KG1112-021-10- | 7-ox0- acid | E.coli BW25113 AdadAX negative 97 heptanoic control using host cells with empty vector KG1012-021-80-3 | 7-Oxo-water acid Negative heptanoic control using water

[00410] Results of biotransformations of m.f-amino acid trans.finase V.f .:

[00411] M-amino V.f transaminase has been successfully demonstrated to have activity in relation to 6-oxo hexenoic acid and 7-oxo heptanoic acid. The formation of the corresponding 6-amino hexenoic acid and 7-amino heptanoic acid products were measured by HPLC. The peak product identification was determined by correlating the retention time of its respective external reference standard.

[00412] It was observed that significantly more 6-amino hexenoic acid was generated under these conditions, providing strong evidence that this transaminase has greater activity in relation to 6-0xo hexenoic acid compared to 7-oxo heptanoic acid.

[00413] In both cases, significant amounts of each product were also observed in the negative control experiment using cells with an empty vector, ie E. coli strains not expressing the target transaminase. We predict that these results are due to the preceding transaminase activity of the free host cell extract. However, the HPLC results clearly demonstrate that for both substrates, higher levels of product were formed in experiments containing cells with the co-expressed transaminase enzyme compared to negative controls. No product formation observed either in the negative control experiments using water as a replacement for cell-free extract. Biotransformation conditions (N-ter-His-Tag) Mw-amino Bd acid transaminase: Concentration of the 10 mM monosodium L-100 mM glutamate substrate (amino donor) 100 ul charge purified N-ter-His-Tag biocatalyst ( 9 / L) 10 ul purified N-ter-His-Tag 1 ul purified N-ter-His-Tag Final volume of 5 mb reaction Biotransformation protocol (N-ter-His-Tag) Mw-amino acid transaminase Bd:

[00414] 14 biotransformations were then performed in a final volume of 1 ml. Each biotransformation comprising 10 mM semialdehyde, 100 mM L-monosodium glutamate and a range of volumes of purified enzyme aliquots (100, 10 and

1 µL). This was done to assess transaminase activity at different protein concentrations.

Each biotransformation was carried out at pH 7 and placed in an incubator with shaking at 30ºC (250 rpm). Sampling was performed periodically and analyzed by HPLC.

Before injection, each sample was heated to 100ºC for 5 minutes, rotated at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter.

List of biotransformation (N-ter-His-Tag) W amino acid transaminase B.d &: Substrate | biocatalyst | Biocatalyst comment volume 1 | Acid 6- | transaminase del 100 uu Aliquot used of the oxo-acid M-amino purified enzyme N-hexanoic B.OM ter-His-Tag 2 | Acid 6- | E. coli (BL21 100 UL OxXxo- DE3 negative control) using hexanoic host cells with empty vector.

Undergoing the same purification process as the superexpression strain 3 | Acid 6- Water 100 UL Oxo-negative control using water instead of hexanoic cells 10-UL transaminase 10 UL oxo-oxo M-amino hexanoic acid B.O [Acid 6- | E. coli (BL21 10 UL oxXxo- DE3) hexanoic acid 6- | transaminase 1 ul oxo-M-amino hexanoic acid B.OM ma oxo- DE3) hexanoic acid 7- | transaminase del 100 uL Aliquot used of the oxo-acid M-amino purified enzyme N-heptanoic | B.The ter-His-Tag 9 [Acid 7- | E. coli (BL21 100 UL OxXxo- DE3 negative control) using heptanoic host cells with empty vector.

Subjected to the same purification process as the super express strain 10 | Acid 7- water 100 UuL Negative oxXxo- control using water instead of heptanoic) from cells

11 | Acid 7- | 10 ul transaminase Mw-amino heptanoic acid B.OM 12] Acid 7- | E. coli (BL21 10 ul oxXxo- DES3) heptanoic 13 | 7- | transaminase acid of 1 u1º oxo Mm-amino heptanoic acid B.O 14 | Acid 7- | E. coli (BL21 1 UL oxo-DE3) heptanoic) Results of biotransformation of M-amino B.dA transaminase biotransformation

[00415] M-amino B.Mm N-ter-His-Tag acid transaminase has been shown to have low activity compared to 6-oxo hexenoic acid and 7-oxo-heptanoic acid. The HPLC results demonstrate that the desired amino acid products were formed in experiments where 100 UL of the purified enzyme was loaded.

[00416] There seems to be no significant difference between the substrate specificity of C6 and C7, when the product levels measured in each experiment were compared. However, in both cases, the level of the product formed was very low.

[00417] In order to ensure the correct identification of the product peaks at a low level in the related chromatograms, a series of samples preach the use of appropriate external reference standards were performed. In each case, a clear increase in the peak product size was observed. The volume folds an external reference pattern was proportional to increase at the observed peak amplitude. HPLC analytical conditions:

[00418] Instrument: Agilent 1200; Column: GraceSmart RPC18

150 x 4.6 mm, 5 microns; Column temperature: 40ºC, flow rate: 1 mL / minute; Detection: UV 338 nm; injection volume: 14 ul, mobile phase: Line A: 5 mM sodium phosphate; pH 7; Line B: acetonitrile. Gradient: Rr | 6 | 8 | PB and | == Derivatisation of non-chiral HPLC pre-column was performed as follows, for each sample that was taken:

[00419] A 5 mL solution containing B-mercaptoethanol (50 UuL), o-phthalialdehyde (50 mg), 400 mM potassium borate buffer, pH 10 (4.5 mL) and methanol (0.5 mL) was prepared. Derivatisation was performed on the HPLC auto-sampler using the injector program below; 2 UL of sample, 6 UML of derivatisation mixture, 6 UuL of potassium borate buffer, pH10, was mixed in the injector handle and held for 4 minutes before injection.

[00420] Standard reference concentrations: 10 mM 6-amino capranoic acid; 10 mM 7-amino hexenoic acid; 50 mM of L-monosodium glutamate.

[00421] Conclusion: the conversion of semialdehyde from adipic acid and semialdehyde from pyelic acid to the corresponding 6-amino hexenoic acid and 7-amino heptanoic acid was measured by HPLC. The methyl semialdehyde esters were chemically prepared, the final stage of synthesis was hydrolysis of the appropriate methyl ester using an immobilized lipase giving rise to a semialdehyde solution which was used without further purification. Product formation (W-amino acid) was observed for both c6 and C7 semialdehyde using the Omega aminotransferases from both, Bacillus spahericus D-AAAT (WT gene: U26732.1 (fragment 427.1278) WT protein: P54693.1) as an example of a D-selective alpha-amino acid aminotransferase to demonstrate the conversion of Qa-keto suberate to Qa-ramino-suberate. General procedure for preparing 2-oxo acid substrates: o HO. Eto n OH -> n OEt Oo 1 o 2 n = 1 adipic acid n = 1 diethyl adipate n = 2 pyelic acid n = 2 diethyl pimelate o Eto Ho. O-Nat <—— n OEt n Oo o Oo oo OEt + 3 n = 1 monosodium salt of 2-0x0o pimelate n = 1 adipate of n = 2 monosodium salt of 2-0x suberate triethyl oxalyl n = 2 triethyl oxalyl pimelate Reference literature: J. Org. Chem. 1986, 51, 2389-2391 and Organic syntheses, Coll. Vol. 3, p510, 1955 Preparation of diethyl ester (2):

[00422] A RB flask containing absolute ethanol (880 mL) was cooled to 0ºC. Acetyl chloride (124 mL, 1.71 mol, 7 equiv.) Was added dropwise over 1 hour. Dioic acid

(1) (0.245 mol, 1 equiv.) Was loaded and the resulting suspension was allowed to warm to RT. Once the reaction was completed, as determined by thin layer chromatography, water (300 ml) was charged and the ethanol was removed by evaporation. The remaining aqueous solution was neutralized to pH 7 with saturated sodium bicarbonate solution. The product was extracted with methyl tert-butyl ether (3 x 250 ml), dried over sodium sulfate, filtered and concentrated in vacuo to obtain the crude diethyl ester (2) as a colored oil in an isolated yield of 70% . The diethyl ester was purified by high vacuum distillation before the next stage.

Preparation of the triethyl oxalyl intermediate (3):

[00423] In a flame-dried flask containing anhydrous diethyl ether (161 mL) was loaded with lumps of potassium metals (89, 206 mmol, 1 equiv.) Under an atmosphere of nitrogen. Anhydrous ethanol (32 ml, 536 mmol, 2.6 equiv.) Was added dropwise over 30 minutes. Once the potassium metal was completely dissolved (gently heated to 30ºC it was necessary) a pale yellow solution was observed. Diethyl oxalate (27.5 ml, 206 mmol, 1 equiv.) Was added quickly with good stirring. At this point, the intermediate of the diethyl ester (2) (206 mmol, 1 equiv.) In diethyl ether (50 ml) was added over 10 minutes. An orange suspension formed during the addition, which was cooled on ice to 0ºC. After 2 hours, the solids were filtered and the washed filter cake was made using diethyl ether (5x 50 ml). The filter cake (1st harvest) was collected and the filtrate was stored at 4ºC overnight. The additional precipitated solid overnight (2nd harvest). This was filtered and combined with the 1st crop to provide a yellow solid (33.7 g). This was suspended in 5M hydrochloric acid and extractions of diethyl ether (3 x 100 ml) were performed. The combined organics were washed with brine (1 x 100 mL), dried over magnesium sulfate, filtered and concentrated to provide the triethyl oxalyl intermediate (3) as a crude yellow oil (24.5 g).

[00424] Isolation and hydrolysis of the product (4):

[00425] For the crude triethyl oxalyl intermediate (3) (24.5 g) it was dissolved in 10M hydrochloric acid (100 mL) and left stirring overnight at RT. The reaction mixture was concentrated in vacuo to provide a sticky orange oil. This was dissolved in acetone (100 ml) and activated carbon (49) was loaded. The suspension was left stirring overnight at RT and filtered through a pad of celite. The filter cake was washed with acetone (5 x 10 ml) and the resulting pale yellow filtrate was concentrated to provide a pale orange oil (20.5 g).

[00426] The product was redissolved in water (10 ml) (pH 0.2) and neutralized at pH 4 with 10 M sodium hydroxide. A pale yellow suspension formed that was cooled to 0ºC. the isopropyl alcohol (10mML) was loaded with stirring. The resulting suspension was filtered to provide a yellowish-white filter cake (7.4 gq) which was transferred to a vacuum dissector and dried overnight. After this time, the product was isolated as a white-yellow solid (3.77 g). NMR was used to characterize the final product (see section of annexes for spectrum). Biotransformation conditions: IlvE (L-aminotransferase-

selective) Laboratory book reference RH0912-021-56 Concentration of the substrate L-monosodium glutamate 50 mM (amino donor) Biocatalyst load (9 / L) 100 g / L of complete cells (I1vE) Temperature (degree C) final reaction bs D-AAAT (D-selective aminotransferase) Laboratory book reference KG1012-021-59 (2-oxo-suberate) KG1012-021-93 (2-oxo-suberate) D-alanine substrate concentration (amino donor) Co -pyridoxal phosphate factor Biocatalyst load (9 / L) with | 21 g / L of base equivalents in complete whole cell (WCE) cell equivalents Temperature (grade C) Final reaction volume Biotransformation protocols: IlvE (L-AAAT)

[00427] 4 biotransformations were performed each in a final volume of 5 ml. Each biotransformation comprised 10 mM and 50 mM L-monosodium glutamate substrates. The biocatalyst was loaded as complete cells to result in a final concentration of 100g9 / L. The reactions were carried out at 30ºC in 15 ml Falcon tubes with agitation. Each biotransformation was sampled periodically and analyzed by HPLC. Before injection, each sample was heated to 100ºC for 5 minutes, stirred at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter.

Biocatalyst Substrate ID | Comment Experiment RH0912-56-1 | 2-oxo-pimelate | 1 full-cell IL0912-56-2 | 2-oxo-suberate | Complete cell ilvE RH0912-56-3 | 2-oxo-pimelate Water Negative control RH0912-56-4 | 2-oxo-suberate Water Negative control B.s. D-AAAT:

[00428] 9 biotransformations were performed in a final volume of 5 mL. Each biotransformation comprised 10 mM substrates, 50 mM D-alanine and 0.4 mM pyridoxal phosphate. The biocatalyst was loaded as a cell-free extract to obtain a final concentration of 29 / L of total cell equivalents. The reactions were carried out at 30ºC in 15 mL Falcon tubes in a shaking incubator. Each biotransformation was sampled periodically and analyzed by HPLC. Before injection, each sample was heated to 100ºC for 5 minutes, stirred at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter.

Biocatalyst Substrate ID Comment Experiment KG1012-59-1 | Acid 2-0x0o- | B.s. DAAAT extract | Glutaric cell-free positive control KG1012-59-2 | 2-0x0- 117 acid empty vector Negative glutaric control KG1012-59-3 | Acid 2-oxo-Water Negative glutaric control KG1012-59-4 [2-oxo-suberate B.s. D-AAAT (sodium salt) | cell free extract KG1012-59-5 [2-oxo-suberate | 117 empty vector Negative control (sodium salt) KG1012-59-6 | 2-oxo-suberate water Negative control (sodium salt) KG1012-93-1 | 2-oxo-pimelate | Bs D-AAAT extract (sodium salt ) cell-free KG1012-93-2 | 2-oxo-pimelate | 117 empty vector Negative control (sodium salt) KG1012-93-3 [2-oxo-pimelate Negative water control (sodium salt) Conclusion:

[00429] Using E. coli IlvE successfully demonstrated that the L-2-amino submeric acid and L-2-amino pyelic acid products were generated from 2-oxo suberate and 2-oxo pimelate.

[00430] The results of biotransformation were determined by HPLC correlation in the retention times of the external reference standard for the observed product peaks formed in each biotransformation.

[00431] A non-chiral derivatisation HPLC method was employed to enable the detection of the adducts of the corresponding derivative product at 338 nm in a reverse phase in column C18. IlvE is well known to be enantiospecific by forming only L-amino acids from keto acids so that an analytical achiral method was used for simplicity. Details for the non-chiral derivatisation protocol are highlighted in the attached section.

[00432] It appears that the activity of the enzyme in relation to the L-2-amino / pimelate 2-0oxo pyelic acid measured by HPLC is considerably less than the L-2-amino / 2-oxo suberate submeric acid under these conditions. To investigate this additional surprising result, biotransformation was repeated using a different batch of 2-0x pimelate, however the same result was obtained, suggesting that the activity with respect to compound C8 is actually greater than with respect to c7 opposites. This is particularly surprising as a result of that C5 amino-diacid, glutamic acid, is a common amino donor for all these reactions.

[00433] The negative control experiments were conducted by replacing the entire cell with water. No previous activity was observed for both substrates in these experiments.

[00434] Using Bacillus sphaericus D-AAAT it was successfully demonstrated that D-2-amino glutamic acid (positive control) and D-2-amino submeric acid and D-2-amino pyelic acid products were generated at from 2-oxo-glutarate, 2-0x0 suberate and 2-oxo-pimelate respectively.

[00435] The results of the biotransformations were determined by the retention time of the HPLC of correlation of the external reference standards for the peak of the observed product formed in each biotransformation.

[00436] In addition, HPLC was also employed to demonstrate the enantioselectivity of the enzyme.

[00437] Using a pre-column derivatisation protocol for each sample in the presence of a chiral derivatisation reagent, it was possible to separate the corresponding diastereomeric adducts in a reverse phase in a C18 column at 338 nm wavelength. Details for the chiral derivatisation protocol are highlighted in the attached section.

[00438] Two negative control experiments were run for each substrate; 117 of empty vector cells, that is, the E. coli strain not expressing the target aminotransferase and water.

[00439] A small amount of prior conversion was observed in negative control experiments with empty vector I17 for each substrate. We believe that this result is due to the low level of antecedent aminotransferase activity in the free host cell extract. This is most likely a combination of endogenous alanine racemase activity generating some L-alanine that subsequently act as amino acid donors for endogenous L-AAAT enzymes in E. coli.

[00440] No detectable levels of the expected amino acids were observed in any of the negative control experiments in water under these conditions. HPLC analytical conditions:

[00441] Instrument: Agilent 1100; Column: Phenomenex Kinetex 150 x 4.6 mm, 5 micro GraceSmart 150 x 4.6 mM, 5 microns; Column temperature: 40ºC, flow rate: 1 mL / minute; Detection: UV 338 nm; injection volume: 14 ul, mobile phase: Line A: 5 mM sodium phosphate; pH 7; Line B: acetonitrile. Gradient:

Rr Pee | PB and | == = Derivatisation of non-chiral HPLC pre-column was performed as follows, for IlvE experiments:

[00442] A 5 ml solution containing B-mercaptoethanol (50 UuL), o-phthalialdehyde (50 mg), 400 mM potassium borate buffer, pH 10 (4.5 ml) and methanol (0.5 ml) was prepared. Derivatisation was performed on the HPLC auto-sampler using the injector program below; 2 UL of sample, 6 UML of derivatisation mixture, 6 UL of potassium borate buffer, pH10, was mixed in the injector handle and held for 4 minutes before injection.

Derivatisation of chiral HPLC pre-column was performed as follows for D-AAAT experiments:

[00443] A 5 ml solution containing Boc-L-cysteine (128 mg), o-phthalialdehyde (50 mg), 400 mM potassium borate buffer, pH 10 (4.5 ml) and methanol (0.5 mL) was prepared. Derivatisation was performed on the HPLC auto-sampler using the injector program below; 2 UL of sample, 6 ML of derivatisation mixture, 6 UL of potassium borate buffer, pH10, was mixed in the injector handle and held for 4 minutes before injection.

[00444] Standard reference concentrations:

[00445] 10 mM D-glutamic acid; 10 mM DL-2-amino submeric acid; 10 mM DL-2-amino pyelic acid; 10 mM of subterranean L-2-amino acid; 50 mM D-alanine; 50 mM L-MSG.

3.1.7 - Conversion of sub-a-amino acid to 7-amino heptanoic acid by QO-amino decarboxylases (EC 4.1.1.- (See, figure 10).

[00446] The appropriate catalysts have been selected from a-amino acid decarboxylases as candidates for the decarboxylation of subterranean OQO-amino acid, in particular 4-decarboxylase aspartate (EC 4.1.1.11), glutamate decarboxylase (EC 4.1 .1.15), lysine decarboxylase (EC 4.1.1.18), diaminopimelate decarboxylase (EC 4,1.1.20), and diaminobutyrate decarboxylase (EC 4.1.1.86).

[00447] Escherichia coli GadA glutamate decarboxylase and LysA diaminopimelate decarboxylase in Escherichia coli were initially selected from these protein groups. Other suitable enzymes include LysA from Methanocaldococcus jannaschii (aab99100) (EC 4.1.1.20) (Ray, SS, et al., “Cocrystal structures of diaminopimelato decaroxylase: mechanism, evolution, and inhibition of na antibiotic resistance acessory factor. Structure, 2002. 10 (11): p. 1499-508), and the lysine decarboxylase present in E. coli: CadA (AAAZ23536), which is inducible under anaerobic and lysine conditions, and has a much greater activity and Lde (access D87518) which is constitutively expressed but has less activity for lysine (Kikuchi, Y., et al., “Characterization of a second lysine decarboxylase isolated from Escherichia coli., - J. Bacteriol.,” 1997, 179 914): p. 4486-9).

[00448] The DNA sequences corresponding to the selected genes as well as their related protein sequence are shown in SEQ ID Nos: 15-18. The Escherichia coli LysA decarboxylase decarboxylase sequence was selected as an E. coli codon optimized gene as previously described (“Preparation of 6-aminocaproic acid from 5-formyl valeric acid: DSM- US patent 2011-0171699 Al). Escherichia coli GadA glutamate decarboxylase was selected as the allele-wild sequence. The genes were cloned and expressed using methods described in previous examples.

[00449] In addition to the previously prepared constructs (I29: pET21-GadA and I30: pET21-LysA, 2 additional constructs were prepared by introducing the ilvE fragment (arrays 131 and 132 below) using inABLE technology. Nter amino acids of the branched acid aminotransferase in E. coli (IlvE) upstream of the protein of interest has previously been shown to result in an increase in the expression of some target proteins in E. coli. Sequence 129 E. coli GadA WE) M84024. 1 130 E. coli LysA (codon JA1l14145.1 optimized for E. coli) 131 E. coli GadA iLvE First 45 bases of the iLvE gene were merged into the GadA 132 gene E. coli LysA iLvE First 45 bases of the iLvE gene were merged into the gene LysA iLvE (allele-X02413.1 sequence (wild-allele sequence) wild) fragment 301-1230 The expression results are summarized below: Soluble fraction | Insoluble fraction E: coli Gadà E. coli LysA E. coli GadA ilvE E. coli LysA LlvE Preparation of extracts of to evaluate the activity:

[00450] Frozen cell pellets previously obtained from 24-hour post-induction samples were resuspended in bead lysis buffer consisting of 0.1 mM Tris, 1 mg / ml Pepstatin A and 200 mM PMSF protease inhibitors (161 UL of the mixture should be used for the lysis of the cell pellet of 1 ml of culture in an OD 'o'o of 5), supplemented with 1.5 mg of lysozyme. Acid wash of 212-300 µm glass tubes (0.4 gq per 1 ml of lysis buffer) were added to each sample. Lysis was performed for 30 seconds vortexing at full speed, followed by 30 seconds of incubation on ice, repeated in a total time of 10 minutes.

[00451] The 1 ml samples were taken and remained in the culture used to evaluate the activity. The 1 mL samples were centrifuged at 13000 rpm for 2 minutes and the supernatant separated. SDS samples were prepared and analyzed on SDS-PAGE gel as described above (Figure 18). Following lysis, there is a large band in the soluble fraction for the E. coli GadA ilvE protein (131) (figure 18, Column 4) that was not present in the 1 mL sample, lysed by “Bugbuster” (“insect destroyer” ”). The E. coli LysA protein (132), which previously demonstrated high soluble expression, did not show much soluble expression in the lysis of the beads (Figure 18, Column 5). This can indicate a great variability in this method of lysis. The crude extracts were then used to monitor the activity of proteins expressed from 4 constructs 129 to 132.

[00452] For biotransformation reactions, large-scale cultures were performed and free cell extracts containing 20g9 / L of all cell equivalent were prepared. The host cells carrying the empty vectors were used as negative controls.

[00453] Biotransformations were performed to evaluate the activity for 2-amino suberate as well as for 2-aminoheptanoic acid. Each biotransformation comprised 10 mM of substrate, 10 µg / ml of pyridoxal phosphate and 1 mM of b-mercaptoethanol, adjusted to a pH of 7 with 1M of sodium hydroxide. The biocatalyst was loaded as the cell-free extract to obtain a final concentration of 20 g9 / L of cell equivalents. The reactions were carried out at 37º Cc in 15 ml Falcon tubes with agitation. Each biotransformation was sampled periodically and analyzed by HPLC. Before injection, each sample was heated to 100ºC for 5 minutes, rotated at 14000 rpm for 2 minutes and filtered through a 0.2 micron syringe filter. the o

OH NA or EA, inADIOW NH, 9 n = 1 DL-2-aminopimelic acid n = 1 6-amino capranoic acid n = 2 DL-2-aminosuberic acid n = 2 7-amino heptanoic acid The reaction mixtures were analyzed by HPLC as follows:

[00454] Instrument: Agilent 1100; Column: GraceSmart RPC18 150 x 4.6 mm, 5 microns; Column temperature: 40ºC, flow rate: 1 mL / minute; Detection: UV 338 nm; injection volume: 14 ul, mobile phase: Line A: 5 mM sodium phosphate; pH 7; Line B: acetonitrile. Gradient: PL o | 5 L a | 25 | | 6 | so | | 8 | so | =

Derivatisation of non-chiral HPLC pre-column was performed as follows, for each sample that was taken:

[00455] A 5 ml solution containing B-mercaptoethanol (50 UuL), o-phthalialdehyde (50 mg), 400 mM potassium borate buffer, pH 10 (4.5 ml) and methanol (0.5 ml) was prepared. Derivatisation was performed on the HPLC auto-sampler using the injector program below; 2 UL of sample, 6 UML of derivatisation mixture, 6 UL of potassium borate buffer (pH10), was mixed in the injector handle and held for 4 minutes before injection.

[00456] Standard reference concentrations: 10 mM 6-amino hexanoic acid; 10 mM 7-amino heptanoic acid; 10 mM DL-2-amino submeric acid; and 10 mM DL-2-pyramid amino.

[00457] It was successfully demonstrated that all a-amino acid decarboxylases had activity in relation to 2-amino pyramic acid and 2-submeric acid. The formation of the corresponding 6-amino hexenoic acid and 7-amino heptanoic acid products were measured by HPLC. The peak product identification was determined by correlating the retention time of their respective external reference standards.

[00458] In experiments that evaluated strains 129, 130 and 131, a lower level of activity was observed for both substrates. A number of samples have been fortified with the corresponding external reference standard to ensure that the correct peak has been identified.

[00459] For both substrates, I32 E. coli Lysa IlIvE gave the best results, showing greater activity compared to the other strains.

[00460] In addition, very low levels of each product were also observed in the negative control experiments using empty vector cells, that is, E. coli strain not expressing the target decarboxylase. We predicted that these results were due to previous decarboxylase activity in free host cell extracts.

[00461] However, the HPLC results clearly demonstrate that higher levels of the products were formed in experiments containing cells with the super-expressed decarboxylase enzyme compared to negative controls. No product formation was observed even in the negative control experiments using water as a substitute for the cell-free extract.

[00462] The observed peak concentration corresponding to 7-amino heptanoic acid is shown in figure 19.

3.1.8 - conversion of submeric a-keto acid into pyelic acid semialdehyde by a-keto decarboxylases (EC 4.1.1.-):

3.1.8.1 - Selection of appropriate catalysts:

[00463] Among the decarboxylase family, a-keto acid decarboxylases have emerged as the best candidates for the decarboxylation of submerged a-keto acid, in particular, the pyruvate decarboxylase (EC 4.1.1.1) benzoyl decarboxylase format (EC 4.1. 1.7) and branched-chain 2-oxo-acid decarboxylase (EC

4.1.1.72).

[00464] The protein coding genes for each of these four groups have been identified from a wide range of hosts and their products characterized biochemically and in some cases, structurally (The crystal structure of benzoylformate decarboxylase at 1.6A resolution -Diversity of catalytic residues in ThDP-dependent enzymes: Hasson et al. Biochemistry 1998, 37, 9918-9930; High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis: Dobritzsch et al. J. Biol. Chem. 1998, 273, 20196-20204 Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction: Berthold et al. Acta Crystallographica Sec. D 2007. D63, 1217-1224).

[00465] The following enzymes were then selected among those groups according to their substrate specificity: Lactococcus lactis kivD - 2-ketoisovalerate decarboxylase (GeneBank JAL 14157); Lactococcus lactis kdcA - branched-chain alpha-keto acid decarboxylase (GeneBank JAl 14154), Saccharomyces cerevisiae pdel - pyruvate decarboxylase (GeneBank JAl 14148), Zymomonas mobilis pde (I1472A) mutant of pyrobyldase and pyruvate (15) (A4601I) benzoylformate mutant (GeneBank AY143338; fragment 2860-4446).

[00466] The first four enzymes previously showed some activity in the decarboxylation of acid - pyaric Qar-keto, the C-7 derivative of the target compound, subterranean Qa-keto acid (Preparation of 6-aminocaproic acid from 5-formyl valeric acid: DSM - US201 1-0171699 A1).

[00467] The characterization of the A460I mutant of the Pseudomonas putida benzoylformate decarboxylase demonstrated increased selectivity in relation to hexenoic 2-keto acid (Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzovylformate decarboxylase from Pseudomonas et. Des. Sel. 2005, 18, 345-357) compared to the allele-wild enzyme that demonstrated low activity in relation to 2-keto octanoic acid (Comparative characterization of ThPP-dependent decarboxylases: Gocke et al. J. Mol. Cat. B: Enzymatic 2009, 61, 30-35).

3.1.9 - conversion of submeric a-keto acid into pyelic acid semialdehyde by putative acetolactate synthases with decarboxylase activity:

[00468] The route for synthesis of coenzyme B [(7-hercaptoheptanoil) threonine phosphate] was postulated by White [White RH., (1989) Biosynthesis of the 7-mercaptoheptanoic acid subunit of component B [(7-mercaptoheptanoyl) threonine phosphate ] of methanogenic bacteria, Biochemistry, 28: 860-865] based on the analysis of other metabolic routes in Archea and on the mass spectroscopy analysis of incorporation of 2H and 13C labeled precursors into the final product. The route contains a non-oxidative decarboxylation step of subteric 2-keto acid to 7-oxo heptanoic acid (semialdehyde pimelate) mediated by a postulated 2-keto acid decarboxylase. In the next step, 7-mercapto heptanoic acid is formed. Since this product is formed only (and not C5 and C6 equivalents), this indicates that at least one of the upstream enzymes in the route will accept only C8, but not C6 and C7 substrates. The route was verified to confirm the elongation reaction of 2-keto dicarboxylic acid, from 2-keto glutarate to 2-keto suberate using GC-MS [White RH (1989) A novel biosynthesis of medium chain length alpha-ketodicarboxylic acids in methanogenic archaebacteria. Archivers of Biochemistry and Biophysics, 270: 691-697] and synthesis of 7-mercaptoheptanoic acid and coenzyme B from T7-oxoheptanoic acid [White RH (1989) Steps in the conversion of a-ketosuberate to 7-mercaptoheptanoic acid in methanogenic bacteria, Biochemistry, 28: 9417-9423; White RH (1994) Biosynthesis of 7-mercaptoheptanoylthreonine phosphate, Biochemistry, 33: 7077-7081]. the genes encoding the enzymes responsible for some of the stages of the route (aksA, aksD, aksE and aksF) were identified, cloned and expressed in E. coli, but not the sub-keto acid decarboxylase gene encoding [Howell DM, Harich K, Xu H, White RH. (1998) Alpha-keto acid chain elongation reactions involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in methanogenicArchaea. Biochemistry, 37: 10108-10117; Howell DM, Graupner M, Xu H, White RH. (2000), - Identification of enzymes homologous to isocitrate dehydrogenase that are involved in coenzyme B and leucine biosynthesis in methanoarchaea. J Bacteriol. 182: 5013-5016; Drevland RM, Jia Y, Palmer DR, Graham DE. (2008) Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis. J Biol Chem. 283: 28888-28896]), Nothing is known about the non-oxidative decarboxylation of submeric 2-keto acid in 7-oxoheptanoic acid and this activity has not been reported in vitro. However, 2-ketosuberic acid was converted to coenzyme B by whole cells [White RH (1989)].

Methodology:

[00469] The work done by White and Graham showed that Archaebacteria (such as Methanocaldococcus jannaschii) probably has an enzyme responsible for the non-oxidative decarboxylation of subteric 2-keto acid in 7-oxo heptanoic acid. Other enzymes involved in the coenzyme B route were identified by analyzing the genomes of M. jannaschii [Bult CJ, White O, Olsen GJ, Zhou L, Fleischmann RD, Sutton GG, Blake JA, FitzGerald LM, Clayton RA, Gocayne JD, Kerlavage AR, Dougherty BA, Tomb JF, Adams MD, Reich CI, Overbeek R, Kirkness EF, Weinstock KG, Merrick JM, Glodek A, Scott JL, Geoghagen NS, Venter JC. (1996) Complete genome sequence of the methanogenicarchaeon, Methanococcus jannaschii. Science. 273: 1058-1073] and Methanobacterium thermoautotrophicum AH [Smith DR, Doucette-Stamm LA, Deloughery C., Lee H., Dubois J., Aldredge T., Bashirzadeh R., Blakely D., Cook R., Gilbert K. , Harrison D., Hoang L., Keagle P., Lumm W. Pothier B., Qiu D., Spadafora R., Vicaire R., Wang Y., Wierzbowski J., Gibson R., Jiwani N., Caruso A ., Bush D., Reeve JN, (1997) - “Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics”, J. Bacteriol. 179: 7135-7155]. The genomes were analyzed for the presence of the target gene group and were evaluated in BLAST for proteins showing homology to the existing enzymes. We conducted a similar approach to identify the gene encoding the subteric 2-keto acid decarboxylase. Results: Analysis of gene groups:

[00470] Bacterial genomes can show a level of organization, in which the genes encoding enzymes participating in a rule in the specific route are located in operons, and / or groups of genes. Due to the fact that other enzymes from the BE coenzyme route have been identified and the sequences of some archeabacterial genomes have been published, an attempt was made to identify the sub-keto acid decarboxylase.

The table below shows the genes and enzymes involved in the route:

Enzyme Reaction Methano- Methano- caldococ- | bacterium cus termoauto- jannaschii | tropicum AKSA Qa-keto Synthase (R) condensation - | glutarate and acetyl-CoA; | MJOS503 MTH1630 homocitrate condensation of adipate a-keto and pimelate a-keto in acetyl-CoA

AKsSDAkKSE (R) dehydration -

Homocitrate synthetase and analogs MTH1113 homocitrate / in cis-homoaconitrate MJ01003 MTH1631 dehydratase and or the respective MJ1271 cy-homoaconi- analogues, and the tatehydrase hydration of the products in homoisocitrate and their respective AksF dehydrogenase dehydrogenase dehydrogenase (-) - NAD-isohomocitrate -treo-isocitrate, (-) - treo-it dependent (homo); citrate and (-) - threocide (homo); -carboxylase citrate 2-keto acid decarboxylation | submeric 2-keto acid ?? ?? submerged Mercaptoheptane synthetase ?? ?? act Mercaptoheptane synthetase ?? ?? iltreonin N- (7-mercaptoheptanoyl) -3- 7? 7? O-phosphono-L- threonine

[00471] The coenzyme B biosynthetic genes obtained from M. jannaschii and M. thermoautotrophicum do not appear to reside in the gene groups ("gene clusters"), except for the synthase (R) -homocitrate (MTH1630) and large coding genes homocitrate / dehydratase synthase subunit (MTH1631) in M. thermoautotrophicum. Analysis of operons — hypotetics containing any of the genes listed above did not show the presence of any protein with homology to the known decarboxylases. Therefore, the search was made for decarboxylation analogues in related metabolic routes in M. hannaschii. Genome analysis of Methanocaldococcus jannaschii:

[00472] Analysis of other metabolic routes in M. jannaschii demonstrated that a similar decarboxylation is present in the coenzyme M route [Graupner M., Xu H., and White RH., (2000) - Identification of the gene encoding sulfopyruvate decarboxylase , an enzyme involved in biosynthesis of coenzyme M, = J. Bacteriol., 182: 4862-4867]. o o o o co, MA o MX PV V vi comDeomE = o No FAN, Fosfoenoil- º. 3-sulfopyruvate º Sulfoacetaldehyde Coenzyme M pyruvate

[00473] The non-oxidative decarboxylation of 3-sulfopyruvate in sulfoacetaldehyde is catalyzed by sulfopyruvate decarboxylase (EC 4.1.1.79) containing two subunits, comD (SEQ ID NO: 19) and eats (SEQ ID NO: 20). As both coenzymes B and coenzyme M are involved in methanogenesis and the reactions downstream of decarboxylation are analogous, decarboxylases may develop together and show high homology to each other.

[00474] ComD, ComE and their orthologists found in the BRENDA database were used as keys ("queries") for research in BLAST, Methanocaldococcus jannaschii. The best combination was obtained using Methanocella paludicola ComD / ComE decarboxylase (SEQ ID 21).

[00475] Only two reading opening structures (MJO0277 and MJO663) encoded by the genome of M. jannaschii showed significant homology to comD / comE with the value E <0.0001. Both were noted as acetolactate synthases (SEQ ID 22 and SEQ ID 23).

[00476] Another search was performed using decarboxylase sequences accepting similar substrates that were deposited in the BRENDA database (2-keto glutarate decarboxylase, oxaloacetate decarboxylase, Q & decarboxylase decarylate, transaminated amino acid decarboxylase, benzoyl decarboxylase, decarboxylase 2 - arginine keto, phosphonopyruvate decarboxylase, pyruvate decarboxylase and indole-3-pyruvate decarboxylase).

Key Substrate Source Hits (value E <107) (UNIPROT name & entry) Kgd Descarboxylase | Mycobacterium Without Hits Q9Cc97 2-keto leprae glutarate EC

4.1.1.71 CcitM Descarboxylase | Lactococcus Without Hits Q7TX425 oxaloacetate lactis Kivd Descarboxylase | Lactococcus NP 247250.1 synthase Q68477 a-keto lactis acetolactate isovalerate Catalytic subunit EC4.1.1.1 E-value: 8e-29 Major subunit IlvB5Se-l19 ARO1lO Descarboxylase | Saccharomyces NP 247250.1 synthase Q06408 | amino acid cerevisiae acetolactate transaminated EC catalytic subunit 2e-17

4.1.1.- NP 247647.1 acetolactate synthase large subunit IlvB9e-12 P58416.1 sulfopyruvate decarboxylase - beta subunit 43-04 MdlC Descarboxylase | Sulfolobus Np 247250.1 Q97XR3 synthase | benzoyl format | solfataricus acetolactate subunit EC 4.1.1.7 catalytic 3e-23 NP 247065.1 particle protein recognition signal Srp54 6e-04 2V3C C crystal structure of Srp54-Srpl19-7s.S SrpRna 6e- 04 3NDB B crystal structure of a signal sequence linked to signal 6e-04 arul Descarboxylase | Pseudomonas NP 247250.1 synthase B7V368 | 2-keto ariginia) aerugines acetolactate Catalytic subunit 42-71 NP 247647.1 synthase acetolactate subunit large 2e-38 NP-248425.1 cobyrinic acid Synthase a, c-diamide 4e-05 Fom2 Dcarboxylase - sulfotyruvate carotovora - pyruvate subunit beta 3e-20 subspecies atroseptica P58415.1 decarboxylase

(pectobacteri | sulfopyruvate - subunit an alpha 2e-11 atrosepticum) NP 247516.1 ferredoxinoxidoreductase 2- oxoglutarate - beta sube 2e-05 NP 247250.1 acetolactate synthase catalytic subunit 6e-04 PDC6 Descarboxylase | Saccharomyces NP 247250.1 synthase P26263 cerevisieae pyruvate acetolactate subunit isozyme 3 catalytic 6e-23

4.1.1.1 NP 247647.1 acetolactate synthase large subunit IlvB9e-14 P58416.1 decarboxylase sulfopyruvate beta subunit 6e-04 NP 247310.1 hypothetical protein MJ 0337 9e-04 Ipde Descarboxylase Pantoea NP 247250.1 synthase F2EPZ5 indol-3-ananathisate pyrolate 3-indolate-3-ananathisate NP 247647.1 acetolactate synthase large subunit 7e-l12 NP 247516.1 ferredoxinoxidoreductase 2-oxoglutarate - beta subunit 9e-05

[00477] MJI277 (accession number NP 247250) and MJO0663 (NP 247647) were good hits for almost all keys used to search the genome. The protein alignment using the ClustaIV algorithm demonstrated significant homology between MJ0277 and the comD / comE proteins (Figure 20).

[00478] However, MJO0277 demonstrated an even greater homology for LACLA acetolactate synthetase from Lactococcus lactis (figure 21).

[00479] Analysis of functional domains in LACLA acetolactase synthase, with D / come from Methanocellla paludicola and the hypothetical acetolactate synthase MJ0277 demonstrated that they all had a similar structure containing a thiamine-phosphate binding site (Figure 22). Almost identical results were obtained for the second reading opening structure MJO0O663 by Methanocaldococcus jannaschii.

[00480] The enzyme acetolactate synthase (ALS) (also known as acetohydroxy acid synthase) catalyzes the conversion of pyruvate to α-acetolactate. o o synthase o o PE J S acetolactate J 1 AO AE Dá "= -— - Á NR Son * Fo: o o É OH

[00481] The first step of the reaction is the decarboxylation of pyruvate (which is a 2-keto acid) in an enzyme binding intermediate that is additionally condensed with a second pyruvate molecule resulting in acetolactate. Thus, 2-keto acid decarboxylases and acetolactate synthases are likely to show high homology to each other. Conclusion:

[00482] Analysis of the genome of M. jannaschii demonstrated the presence of two proteins annotated as acetolactate synthases (MJ0277 and MJO663) that can be previously unidentified in the non-oxidative sub-keto acid non-oxidative decarboxylase involved in biosynthesis of coenzyme B. Only Methanogenic spp. it apparently has this second version of acetolactate synthase (MIO663) with, for example, Methanocaldococcus fervens (NC 013156.1) (Locus YP 003128272); Methanotorris igneus KoI 5 (NC 015562.1) (Locus YP 004483786) and Methanocaldococcus infernos ME (NC 014122.1 (Locus YP 003615749) all having proteins with more than 75% positive for MJ0663. No non-methanogenic spp., Had more than 50% positive for MJO0666. The other spp., Had a second gene with even homology

MIO277, Methanocaldococcus fervens (Locus YP 003127480); Methanotorris igneus KoI 5 (Locus YP 004484320) and Methanocaldococcus infernos ME (Locus YP 003615922) and this gene was noted as an acetolactate synthase. This gene had homology for acetolactate synthase in other non-methanogenic organisms.

[00483] It is very likely that one of them is the real acetolactate synthase. This enzymatic activity has been demonstrated in many archaebacteria, and M. jannaschii encodes the subunit regulatory acetolactate synthase, MJO161. However, one of the enzymes may be the unidentified 2-keto acid decarboxylase and was not simply observed. It is likely that the regulatory subunit MJO0l161 or ILVN (Methanocaldococcus jannaschii - Locus NP247129), the small subunit of acetolactate synthase is required for the decarboxylase activity of MJO277 or MJO663. This is a small -190aa subunit that binds the dimer or possibly tetrameter of the AHAS / ILVB holoenzyme in equal numbers. ILVN responds to the concentration of valine by acting as a raw material to prevent overproduction (Barak and Chipman 2012). The ILVB holoenzyme is only 5-15% efficient in the absence of ILVN, the regulatory unit has no activity on itself (M. Vyazmensky, C. Sella, Z. Barak, DM Chipman, - “Isolation and Charcterization of Subunits of Acetohydroxy Acid synthase Isozme III and Reconstitution of the Holoenzime ”, - Biochemistry, 35 (1996), pp. 10339-10346).

Alternatively, submeric 2-keto acid can be decarboxylated by an enzyme responsible for decarboxylation of 3-sulfopyruvate in the coenzyme M route. Therefore, decarboxylase comD / come can also be considered as a target enzyme.

[00485] The proteins annotated as acetolactate synthases MJO0277 (NP 247250) and MJOG663 (NP 247647) have been identified as the main candidates for non-oxidative decarboxylation of adipic 2-keto acid, 2-keto pyelic acid and 2-keric acid. Acetolactate synthases have been reported to catalyze the decarboxylation of 2-keto acid (Atsumi, S. et al., 2009, - “Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutane Synthesis in Escherichia coli.”, - Applied and Environmental Microbiology, 75 (19): 6306-6311). In addition: 3-sulfopyruvate decarboxylase with D / come (P58415 / P58416), Kgd (Q9CC97), citM (Q7X4Z5), kivd (Q6837), AROl1O (Q06408), mdlC (Q97XR3), aruIl (B7V368) fom2 (5) PDC6 (P26263) and IpdC (F2EPZ5) are excellent candidates for the conversion of submeric 2-keto acid to pyelic acid semialdehyde (SEQ ID Nos: 24-40).

3.1.10 - Conversion of pyelic a-keto acid to submerged a-keto acid by lengthening the Qa-keto acid chain (See, figure 10):

[00486] 3.1.10.1 - Selection of the target enzyme gene

[00487] The conversion of pyelic a-keto acid to submeric a-keto acid has been previously reported to be part of the biosynthesis of coenzyme B in methanogenic archaebacteria, such as Methanocaldococcus jannaschii, Methanocaldococcus vannielii, Methanocaldococcus maripaludis, Methanocalhina, Methanocalhina (White, "Arch. Biochem. Biophy.", 270: 691-697 (1989)). The genes encoding the enzymes involved in the multiple cycles of chain elongation from keto glutaric acid in submeric acid, homologous to the enzymes involved in leucine and lysine biosynthesis, were identified from Methanocaldococcus jannaschii and their corresponding characterized proteins (reaction and elongation of the Qa-keto acid chain involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in Howell et al., “Biochemistry” 37: 10108-10117 (1998): Drevland et al., J. Bacteriol. ” , 189: 4391-4400 (2007); Howell et al., "J. Bacteriol.", 182: 5013-5016 (2000); Drevland et al., "J. Biol. Chem.", 283: 28888-28896 92008); and Jeyakanthan et al., “Biochemistyr”, 49: 2687-2696 (2010).

[00488] Orthologous genes have also been identified from Methanobacterium thermoautotrophicum, but their products have not been characterized yet. At least two isogens have been reported in this archaebacterium for each of the required activities (ASKA, AksD / E and AksSF) as previously reported in Methanocaldococcus jannaschii (Smith et al., 1997, 179, 7135-7155). The table below presents the orthology of the corresponding proteins of Methanobacterium thermoautotrophicum with the proteins of Methanocaldococcus jannaschii well characterized based on the previously mentioned literature and their potential function deduced.

Enzyme activity | Proteins | Functions of proteins M. proteins of M. jannaschii orthologues of jannaschii M. termoauto-trophicum AKSA: synthase 2- MJO503 Condensation of isopropylmalate / glutarate a-keto and acetyl-CoA synthase homocitrate) | to form homoaconitate trans-MTH1630; functions in the synthesis of suberate a-keto MJ1195 Formation of 2-isopropylmalate from acetyl-CoA and 2-oxoisovalerate in MTH1481 AksD leucine biosynthesis: dehydratase MJ1003 Formation of cis-homo, 3-7 aconitate (n = 1-4) isopropylmalate / hom | when coupled with MTH1631 oaconitase MJ1271; involved in the (large subunit) production of subterranean aceto acid MJO0499 Involved in leucine biosynthesis MTH1386 AksE: dehydratase MJI1271 Formation of cis-homo, MTHO829 or 3-aconitate isomerase (n = 1-4) contains isopropylmalate / when coupled with homoaconitase sequence MJ1003; involved in | consensus of (subunit a- acid | YLTR carac- small) teristic subterranean keto of homo-conitase MJI1277 Involved in MTH1387: leucine biosynthesis Contains the consensus sequence of YLVM characteristic isopropylmalate isomerase AksF: dehydrogenase in MJ1596 involved production of 3-a-keto acid MTH1388 isopropylmalate / sub-sub-MI0720 idrogenase involved in the homoisocitrate leucine biosynthesis MTHO184

[00489] The identification of the orthologists for the chain-lengthening proteins in Methanobacterium thermoatotrophicum has been reported in the literature from the comparative genomic data between the genomes of Methanocaldococcus jannaschii and Methanobacterium thermoautotrophicum, sequence analysis of the corresponding proteins and evaluation of activity. The potential application of Methanobacterium thermoautotrophicum alternative gene products and their corresponding Methanocaldococcus jannaschii orthologists for the synthesis of alpha-keto pyelic acid has been reported (publication WO 2010-104391 A2). Therefore, eight Methanobacterium thermoautotrophicum proteins were selected based on the example of the reaction and elongation of the chain between pyelic acid and submeric acid, that is, AkKSA 2-isopropylmalate synthase / MTH1630 homocitrate synthase (GeneBank: AEOOO0666.1; fragment 1494539-; 1495714) and MTH1481 (GeneBank AEONOO0666.1; fragment 1337346-1338863); AksD dehydratase 3-isopropylmalate / homoaconitase (large subunit) MTH1386 (GeneBank AEOO00666.1; fraction 1253910-12555) and MTH1631 (GeneBank AEOO00666.1; fragment 1495715-1497001); AkKsSE dehydratase or 3-isopropylmalate / homoaconitase isomerase (small subunit) MTHO829 ((GeneBank AEO0O00666.1; Fragment 752586-753098); and MTH1387 (GeneBank AE000666.1; fragment 1255181-1255669); homoisocitrate dehydrogenases, MTHO184 (GeneBank AE 000666.1; fragment 130396-131391); and MTH 1388 (GeneBank AEOO00666.1; fragment 1255666-1256655).

[00490] The allele-wild genes were selected for the construction of corresponding expression vectors in E.

coli, except for the AksA (1) -MTH1630, AksD (2-MTH1631 and AksF (2) -MTH1388 genes, in which the GTG or TTG start codon was changed to ATG.

[00491] 3.1.10.2 - Cloning of expression vectors containing the target genes:

[00492] The selected target genes of Methanobacterium thermoautotrophicum were then cloned into the main chain of inducible pET21-a IPTG using inABLE technology, as described in detail in Section 3.1.1.2) to produce 19 (M. thermoautotrophicum the AKksA gene (1 ) -MTH1630 in pET21-a), 110 ((M. thermoautotrophicum the AksA gene (2) -MTHI1481 in pET21-a), I1l1 ((M. Thermoautotrophicum the AksD gene (1) - MTH1386 in pET21-a), 112 (M. thermoautotrophicum the AksSD (2) -MTH1631 gene in pET21-a), 113 ((M. thermoautotrophicum the AksE gene (1) -MTHO0O829 in pET21-a), I14 (M. thermoautotrophicum the AksE gene (2) - MTH1387 in pET21-a), 115 (M. thermoautotrophicum the AksF (1) -MTHO0O184 gene in pET21-a), and Il6 (M. thermoautotrophicum the AksF (2) -MTHO01368 gene in pET21-a).

[00493] Briefly, the potential Earl sites in the selected genes and the vector's main strand were disrupted to prevent interference with the partial fusion / fusion preparation involving EarI digestion / ligation cycles. No EarI sites were observed in AksD (1) -MTH1386, AksD (2) - METH1631 and AksE (2) -MTH1387. One Earl site was observed in each of the AKksA (1) -MTH1630, AKksSA (2) -MTH1481, AksSE (1) -MTH0829, and AksF (2) -MTH1388 genes, and two EarI sites were found in the AksF ( 1) -MTHO184, three EarI sites were detected in the main chain of the pET21-a vector. EarI sites were disrupted by introducing a silent mutation within the restriction site. The three EarI sites in the vector were disrupted by mutating the last guanidine base of the restriction site within the thymine.

[00494] EarI free sequences were then used as input sequences in the part design software to construct the flanks of the corresponding truncated parts through the Earl sites, as discussed in Section 3.1.1.2. The synthesized truncated parts of Methanobacterium thermoautotrophicum AksA (1) -MTH1630 (TP480), AKSA (2) -MTH1481 (TP49), AKksD (1) -MTH1386 (TP50), AksD (2) - MTH1631 (TP51), AKsSE (1 ) -MTH0829 (TP52), AKsSE (2) -MTH1387 (TP53), AksF (1) -MTHO184 (TP54), and AksF (2) -MTH1388 (TP55) were inserted into the DNA2.0 pJ20l vector. The truncated part vector pET2l1-a was completely synthesized with the introduction of a fragment including the chloramphenicol resistance gene to produce the vector TP31.

[00495] Phosphorylation and subsequent annealing of inABLE oligonucleotides were performed as described in section 3.1.1.2. Briefly, the oligos were incubated with T4 kinase at 37ºC for 30 minutes, followed by inactivation of the enzyme at 65ºC for 20 minutes. Then, part of the ligation fusions were prepared by ligating the 5 '"end - of the truncated part with its corresponding oligo part of the ring fragment, and the 3'- end of the truncated part with the oligo ligand of the ring fragment, as previously described in section 3.1.1.2 The ligand part fusions corresponding to the selected genes (Methanobacterium thermoautotrophicum - AKkSA (1) -MTH1630, AksA (2) -MTH1481, AksD (1) -MTH1386, AksD (2) -MTH1631, AKkKsE (1 ) - MTHO829, AKksE (2) -MTH1387, AKksSF (1) -MTHO184 and AksF (2) -MTH1388) were prepared by connecting their respective ringed oligo parts (POA48, POA49, POA5SO, POA51l, POAS52, POA5S3, POAS4 , or POAS5S), their truncated parts (TP48, TP49 TP51, TP52, TP53, TP54, or TP55), and the ring ligand oligos from the vector's main chain. by connecting its ringed oligo part, its truncated part, and the oligos li ringed giants from both genes. A gene-free arrangement of the negative control was also prepared by linking the vector main chain of the ringed part oligos, their truncated part, and their ringed oligo ligand, resulting in a self-arrangement of the vector's main chain.

[00496] A ten to 20-fold molar excess of the oligos and ligands to the truncated part was used in the reaction of the gene and vector in favor of the link between the truncated part and the oligonucleotide during each EarI digestion / ligation cycle. The EarI digestion / ligation reaction of 50 UL were incubated in a thermocycler (Eppendorf Mastercycler Gradient), alternating the temperature between 37ºC and 16ºC. Upon completion of the cycles, the samples were loaded onto a 0.7% agarose gel, and the expected size fragments were observed for the preparation of the parts / ligand fusions. The correct size bands were then cut from the gel, and the DNA was extracted on gel using the QIAGEN QIAquick extraction kit. The DNA concentration ranged from 13.6 ng / Ul 24.8 ng / Ul in a total volume of 30Ml (conc. DNA ranges from 4.9 nM to 53.9 nM). The self-arrangement of the fusion of part / ligand (pET21-a part / pET21-a ligand) for the production of the free construct of the negative control gene was also performed.

[00497] The combination of the gene / ligand fusion with its respective vector / ligand fusion was then performed using a 2-part arrangement, as described in section

3.1.1.2. In summary, the gene part ligands and the vector part ligands were incubated at room temperature for 30 minutes before the high-efficiency, chemically competent transformation of NEB10fê E. coli cells using 2141 of the reaction arrangement and 10 41ul of competent cells. The transformed cells were plated on LB-Amp agar, and incubated at 37ºC overnight. About a thousand clones were obtained for each arrangement. Two random clones were selected from each array, and the corresponding vectors were isolated using the QUIAGEN QUIAprep Miniprep kit.

[00498] The restriction was performed to confirm the construction of the vector, as described in section 3.1.1.2. In summary, the clones obtained from 19 and were analyzed using Bg / II and XmnI to identify the positive clones for the insertion of the Methanobacterium thermoautotrophicum AKkSA (1) -MTH1630 gene into pET21-a, the clones obtained from the I10 array were analyzed using PstI and XmalI / XbaIl to identify the insertion of the AksA (2) -MTH-1481 gene into pET21- a, the clones obtained from the Ill array were analyzed using HincIT and AlWNI to identify the insertion of the AksD gene (1) - MTH1386, the clones obtained from the Il12 array were analyzed using NcoI and Bg / II to identify the insertion of the AksD (2) -MTH1631 gene, the clones obtained from the Il13 array were analyzed using MluIl and XmnI to identify the AksE insert (1) -MTH0829 within pET21-a, the clones obtained from the Il14 array were analyzed using PsiI and XmaI / AlwNI to identify the insertion of the AKksE (2) -METH1387 gene into the E. coli expression vector, the clones obtained from the Il5 array were analyzed using AlwNI and HincIIl for i dentify the insertion of the AksF (1) -METHO184 gene into the E. coli expression vector, and the clones obtained from the Il6 array were analyzed using BsalI and HindIII / alwNi to identify the insertion of AksF (2) -MTH1388. The restriction products were run on an agarose gel, and the expected band pattern was observed for the tested clones from each array, confirming the construction of the E. coli pET21-a expression vectors hosting the DNA chain stretching genes. Methanobacterium thermoautotrophicum. In addition, the junction of parts between the 3 '"” end - and the vector's main strand and the 5' ”end - of the genes as well as between the 3 '" end - of the gene and the 5' "end - of the chain main vector were sequenced to confirm the construction.

3.1.10.2 - Expression of exogenous thioesterase genes in E. coli:

[00499] Expression of exogenous chain elongation gene products from Methanobacterium thermoautotrophicum AkSA (1) -MTH1630, AksSA (2) -MTH 1481, AKksSD (1) -MTH1386, AksD (2) - MTH1631, AksE (1) -MTHO829, AksE (2) -MTH1387, AksF (1) -MTHO184 and AksF (2) -MTH0184 in E. coli was performed as described in section 3.1.1.3. In summary, electrocomponent BL21 (DE3) cells were transformed with 10 ng of DNA, and the transformation sample was plated on LB-Amp-Agar. Transformants were obtained after overnight incubation at 37ºC. a single clone was cut from each array, and 5 ml of LB-Amp medium was inoculated as a starting culture. The culture OD600 was measured after overnight incubation at

37ºC and 250 rpm, 2 ml of culture (approximately 8x10 ') were used to inoculate 100 ml of LB-Amp, which was incubated in a baffled flask at 37ºC and 250 rpm.

[00500] Protein expression was induced by the addition of 1 mM IPTG (Final conc.) And cultures were incubated at 37ºC and 250 rpm. A 1 mL sample was taken before induction and after 4 hours and 24 hours of incubation after induction, and OD600 measured to determine cell growth. After 24 hours of post-induction incubation, the remaining culture was transferred to a 50 mL Falcon tube, harvested by centrifugation at 5000 rpm for 10 minutes, and the cell pellets used for further analysis or stored at - 20ºC.

[00501] For SDS-PAGE analysis, the samples were centrifuged at 13000 rpm for 2 minutes, the supernatant was removed, and the cells were lysed using a Bugbuster protein extraction reagent supplemented with lysozyme (15 mg / ML) and benzonase ( 3.4 U / UL). The lysis reaction was then centrifuged at 13000 rpm for 2 minutes, the soluble fraction was transferred to a new tube, and the insoluble fraction was resuspended in water. 20 ul of each fraction were mixed with 80 ul of SDS sample buffer (SDS-load buffer, 9% DTT and water), heated in a heating block for 5 minutes at 95ºC, and 10 ml of each sample preparation- SDS was then loaded onto 4-20% SDS-PAGE tricene gels to analyze the protein content of the soluble and insoluble fractions.

[00502] Only the AksE (2) -MTH1387 protein was successfully expressed in soluble form, that is, an 18 kD band in the soluble fraction after IPTG induction. The AksA (l1) - MTH1630, AkKksD (2) -MTH1631, AksF (1) -MTHO184 and AksF (2) -MTH1388 proteins are expressed in insoluble forms, that is, 44 kD, 36 KkKD and 46 kD bands, respectively , were detected in the insoluble portion. No expression was detected for the AksA (2) -MTH1481, AksD (1) -MTH1386 and AKsSE (2) -MTH proteins

1387. Therefore, expression optimization was performed for AKksSA (1) -MTH1630, AksD (2) -MTH1631, AksF (1) -MTHO0184, AKksSF (2) -MTH1388, AKksSA (2) -MTH1481, AKsD (1 ) -MTH1386 and AKsE (2) - MTH1387 based on reports of successful expression by Methanocaldoccus jannaschii orthologists in the literature. In particular, literature data on the expression of M. jannaschii orthologists revealed a set of different conditions, in particular terms of inductor concentration and induction temperature. The IPTG concentration was varied between 0.1 and 1 mM (initial conditions of 1 mM) and the induction temperature was varied between 16 and 37ºC (initial conditions 37ºC). The same expression protocol and the same sample preparation SDS-PAGE, as described in section 3.1.1.3, was used for solubility optimization experiments.

[00503] The expression level of the AksA (1) -MTH1630 protein was improved after decreasing and 10 times the concentration of IPTG, but the protein remained insoluble; temperature optimization did not affect expression or solubility. No significant improvement in the solubility of AksD (2) -MTH1631 and AksF (2) -MTH1388 through a 10-fold decrease in IPTG concentration, since both proteins were only visible in the insoluble fractions after induction. No significant improvement in solubility was observed for AksD (2) -MTH1631 and AksF (2) -MTH1388 by decreasing the induction temperature to 16ºC since both proteins were only visible in the insoluble fraction after induction. However, the solubility of Aksf (1) -MTHO184 was improved by lowering the induction temperature to 30ºC. Therefore, AksSA (1) -MTH1630 (19), AksD (2) -MTH1631 (112) and AksF (2) -MTH1388 (I16) can be expressed in different hosts, for example, in the eukaryotic system, like Saccharomyces cerevisiae or in a thermophilic bacterium like Bacillus stearothermophilus.

[00504] Since the Methanobacterium thermoautotrophicum gene products AKSA (1) -MTH1630, AKksSA (2) -MTH1481, AksD (2) -MTH1631 and AksE (1) -MTHO0829 were not expressed in an E-soluble form. coli using the pET21-a / IPTG expression system, these genes were cloned for expression using the expression system in Saccharomyces cerevisiae. The expression vector was arranged using the inABLE technology described above, except that the restriction enzyme SapI was used instead of EarI in the preparation of the yeast vector main chain / ligand fusion, comprising the constitutive promoter ADHlp, the terminal of CYC1lt transcription, and the high-copy vector backbone derived from plasmid YEplacl195. Electrocompetent E. coli TOPIO cells were transformed with each part / ligand arrangement and a larger DNA stock was prepared using the QIAGEN Hi-Speed Plasmid Purification Midi KIT (67.4-173.0 nM in 120 µl). The fragment of expected size was observed for the preparation of the yeast vector part / promoter ligand ADH1, despite the use of SapI, after electrophoresis in agarose gel. The correct bands were then cut from the gel, and DNA was extracted from the gel using the QIAGEN extraction kit

QIAquick Gel (14.1-138.1 nM in 30 ul).

[00505] The final constructs were then prepared by sandblasting incubation of 4-part arrays, ie, ADHI1 promoter, each of the Methanobacterium thermoautotrophicum AKksA (1) -MTH1630, AksA (2) -MTH1481, AksD (2 ) -MTH1631 and AKsSE (1) -MTHO829, the CYC1 terminal and the yeast vector main chain, at 37ºC. Then, 10 4ul of highly efficient and chemically competent E. coli NEB1I0ft cells were transformed with 2 UL of the reaction of the array, plated on LB-Kan-agar, and incubated at 37ºC overnight. Between two hundred and two hundred and fifty clones were obtained for the 4-part arrangements. Five random clones were then removed from the plates, and the corresponding vectors were isolated using the QIAGEN QIAprep Miniprep kit.

[00506] The isolated constructs were analyzed using the restriction analysis. A preliminary analysis was performed using PmeIl / SapI to identify any truncated vector contaminants. The clones had no truncated vector contaminants, so they were then analyzed using the restriction enzymes, XhoI / Ndel, BsaIl and Mlufl in order to confirm the insertion of the 4-part / ligand fusions. The expected band pattern was observed in both clones confirming the construction of the expression vector S. cerevisiae hosting the AH1 promoter, the gene of interest, the CYC1l terminal and the yeast vector main chain. Finally, the junctions between the 4 parts were sequenced to confirm the proper arrangement.

[00507] Transformation with S. cerevisiae I24-127 expression vectors can be performed as described here, and expression from the constitutive promoter ADHlp can be monitored after cell lysis using SDS-PAGE. Construct 124 includes the ADH1 promoter, the Methanobacterium thermoautotrophicum AKsA (1) -MTH1630 gene, and the truncated CYCl terminal inserted into the yeast vector main chain. Construct 125 includes the ADH1 promoter, the Methanobacterium thermoautotrophicum AKksA (2) -MTH1481 gene, and the CYCl terminal. Construct 126 includes the ADH1 promoter, the Methanobacterium thermoautotrophicum AksD (2) -MTH1631 gene, and the CYCl terminal. Construct 127 includes the ADH1l promoter, the Methanobacterium thermoautotrophicum AKsE (1) -MTH0829 gene, and the CYC1 terminal.

[00508] Patents, patent applications, publications, product descriptions, and protocols cited during the description of this specification are incorporated herein by reference in their entirety and for all purposes.

Claims (1)

CLAIMS l. Method for producing a di- or trifunctional alkane, the di- or trifunctional alkane selected from the group consisting of pyelic acid (PA), pyelic acid semialdehyde (PAS), armamino-suberate (AAS), and 1,7-diaminoheptane ( 1,7 DAH), said method, characterized by the fact that it comprises: a) contacting pimeloyl-CoA (PCoA) or pimeloil [acp] (PACP) with an enzyme that catalyzes the hydrolysis of the thioester of PCOA or PACP to pyelic acid (PA ), so that PA is produced, the enzyme being a thioesterase, an acid-thiol ligase, or a CoA transferase; and the thioesterase being one or more of: - the tesB or acotI3 gene product; and - a thioesterase in EC. 3.1.14; the acid thiol ligase being an EC acyl- [acp] synthase 6.2.1.14 (such as BioW) or EC 6.2.1.20; and the CoA transferase being one or more of: - a CoA transferase in EC. 2.8.3.14; and - the thnH gene product; b) contact PCoA or PACP with an enzyme that catalyzes the reduction of PCoOA or PACP to PAS, so that PA is produced, the enzyme that catalyzes the reduction of PCoA or PACP to PAS is an acyl- [acp] -reductase fatty or an aldehyde dehydrogenase in EC 1.2.1.3, EC 1.2.1.20, EC 1.2.1.22, EC 1.2.1.57, or EC 1.2.1.76; Cc) contact PCoA or PACP with an enzyme that catalyzes the reduction of PCoA or PACP to PAS, so that PAS is produced, and the enzyme that catalyzes the reduction of PCoA or PACP to PAS is an acyl- [acp] -reductase fatty or an aldehyde dehydrogenase in EC 1.2.1.3, EC 1.2.1.20, EC 1.2.1.22, EC 1.2.1.57, or EC 1.2.1.76; d) contact QMa-keto suberate (AKS) with an enzyme that catalyzes the AKS keto decarboxylation in PAS, so that PAS is produced, the enzyme being an acetolactate synthase; e) contacting AKS with an enzyme that catalyzes the transfer of an amino group to AKS to produce a-amino suberate (AAS), so that AAS is produced, the enzyme being an aminotransferase in EC 2.6.1.21; f) contact 7 AHT with an enzyme that catalyzes the transfer of an amino group to 7 AHT to produce 1,7-diaminoheptane (1.7 DAH), so that 1.7 DAH is produced, the enzyme that catalyzes the transferring an amino group to the 7 AHT to produce 1,7-diaminoheptane (1.7 DAH) be one or more of: - an amino acid aminotransferase in EC 2.6.1.21; - a deamination dehydrogenase, and - a Qtransaminase in EC 2.6.1.11, EC 2.6.1.13, EC 2.6.1.48 or EC 2.6.1.62; Or g) a capable cell defined in any of steps a) to f) of the process, wherein said cell is an isolated cell comprising one or more exogenous nucleic acid encoding: (i) a thioesterase, an acid-thiol ligase, or a CoA transferase; and (ii) a NAD-dependent non-acylating aldehyde dehydrogenase; (ii) a fatty acyl- [acp] reductase; and (ii) an enzyme that catalyzes a reaction whose product is PCoA or PACP; (iii) an acetolactate synthase; and (ii) and the enzyme that catalyzes a reaction whose product is AKS; (iv) an aminotransferase; and (ii) an acetolactate synthase; or (v) a deamination dehydrogenase; and (ii) an enzyme that catalyzes a reaction whose product is 7 AHT. Optionally, the cell is a prokaryotic cell or a eukaryotic cell. 2. Method according to claim 1, characterized in that the PCoA or PACP is produced through a process comprising one or more steps of: (a) a route shown in figure 10A or Figure 10B; (b) a biotin I biosynthesis route (see figure 6); (ec) a biotin II biosynthesis route (see figure 5); (d) the benzoate or cyclohexane carboxylate degradation route (see figure 7); (e) the cyclohexane carboxylate route (see figure 9); and (f) the condensation of three malonyl-CoA molecules (see figure 4), where PCoA or PACP is produced. 3. Method, according to claim 1 or 2, characterized by the fact that it comprises - additionally, contacting PA with an enzyme that catalyzes the reduction of carboxylic acid from PA to semialdehyde pyelic acid (PAS), where PASS is produced. 4, Method according to claim 3, characterized in that the enzyme that catalyzes the reduction of PA carboxylic acid to PAS is one or more of: (a) a NAD-dependent non-acylating aldehyde dehydrogenase, (b) the shingomonas thnG gene product; and (ce) the product of the Cupriavidus spp. 5. Method, according to claim 1, characterized in that the PCoOA or PACP is produced through a process comprising one or more steps of: (a) a route shown in figure 10A or Figure 10B; (b) a biotin I biosynthesis route (see figure 6); (c) a biotin II biosynthesis route (see figure 5); (d) the benzoate or cyclohexane carboxylate degradation route (see figure 7); (e) the cyclohexane carboxylate route (see figure 9); and (f) the condensation of three malonyl-CoA molecules (see figure 4), where PCOA or PACP is produced. 6. Method, according to claim 1, and relating to the AKS used in step (d) of said main claim, characterized in that said AKS is produced by a process comprising one or more steps of: (a) a route shown in figure 10; or (b) the coenzyme B route of methanogenic arche (see figure 3). 7. Method, according to claim 1, and relating to the AKS used in step (e) of said main claim, characterized in that said AKS is produced by a process comprising one or more steps of: (a) a route shown in figure 10; or (b) the coenzyme B route of methanogenic arche (see figure 3). 8. Method according to claim 1 or 7, characterized in that it comprises - additionally contacting AAS with an enzyme that catalyzes the decarboxylation of AAS amino acid to 7-amino-heptanoic (7AHA) where 7AHA is produced . 9. Method according to claim 8, characterized in that the enzyme that catalyzes the A-amino acid decarboxylation of AAS in 7-amino-heptanoic acid (7AHA) is one or more of a QO-keto-decarboxylase or a acetolactate synthase, or an enzyme in EC 4.1.1.-. 10. Method according to claim 8 or 9, characterized in that it additionally comprises contacting 7AHA with an enzyme that catalyzes the hydrolysis of the amide from 7AHA to enantolactam (ENTL), where ENTL is produced. 11. Method according to claim 8 or 9, characterized in that it comprises - additionally contacting 7AHA with an enzyme that catalyzes the dehydrogenation of 7AHA aldehyde to 7-amino-heptanal (7 AHT), where 7 AHT is produced . 12. Method according to claim 11, characterized in that it additionally comprises contacting 7AHT with an enzyme that catalyzes the transfer of an amino group to 7AHT to produce 1,7-diaminoheptane (1.7 DAH), where 1 , 7 DAH is produced. 13. Method according to claim 1, characterized in that 7 AHT is produced by the dehydrogenation aldehyde of 7AHA, which is produced by one or more stages of a route shown in figure 10. 14. Method according to any one of claims 1 to 4, characterized in that the enzyme, or one or more enzymes, is or is: (1) isolated; (ii) in a cell lysate or in a partially purified cell lysate; or (iii) in his / her recombinant host cell expression. 15. Method according to claim 1 or 5, characterized in that the enzyme, or one or more enzymes, is or is: (1) isolated; (11) in a cell lysate, a partially purified cell lysate, cell lysates, or partially purified cell lysates; or (iii) in one or more expression of his or their recombinant host cell chains. 16. Method according to claim 1 or 6, characterized in that the enzyme, or one or more enzymes, is or is: (i) isolated; (ii) in a cell lysate or in a partially purified cell lysate; or (ii1) in his / her recombinant host cell expression. 17. Method according to any one of claims 1 or 7 to 12, characterized in that the enzyme, or one or more enzymes, is or is: (1) isolated; (11) in a cell lysate or in a partially purified cell lysate; or (iii) in his / her recombinant host cell expression. 18. Method according to claim 1 or 13, characterized in that the enzyme, or one or more enzymes, is or is: (i) isolated; (ii) in cell lysate, or in a partially purified cell lysate; or (iii) in his or their recombinant cell expression.