+

WO2009046929A2 - Biotechnological fixing of carbon dioxide - Google Patents

Biotechnological fixing of carbon dioxide Download PDF

Info

Publication number
WO2009046929A2
WO2009046929A2 PCT/EP2008/008351 EP2008008351W WO2009046929A2 WO 2009046929 A2 WO2009046929 A2 WO 2009046929A2 EP 2008008351 W EP2008008351 W EP 2008008351W WO 2009046929 A2 WO2009046929 A2 WO 2009046929A2
Authority
WO
WIPO (PCT)
Prior art keywords
activity
sequence seq
ncbi
seq
nucleic acid
Prior art date
Application number
PCT/EP2008/008351
Other languages
German (de)
French (fr)
Other versions
WO2009046929A8 (en
WO2009046929A3 (en
Inventor
Joachim W. Schmid
Klaus Mauch
Original Assignee
Insilico Biotechnology Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Insilico Biotechnology Ag filed Critical Insilico Biotechnology Ag
Publication of WO2009046929A2 publication Critical patent/WO2009046929A2/en
Publication of WO2009046929A3 publication Critical patent/WO2009046929A3/en
Publication of WO2009046929A8 publication Critical patent/WO2009046929A8/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

Definitions

  • the invention relates to the biotechnological use of anorgani- see electron donors, especially of gaseous hydrogen for fixing carbon dioxide as a carbon source for the synthesis of organic compounds as energy sources and value products by fermentation in microorganisms.
  • Ralstonia metallidurans Ralstonia metallidurans (Ralstonia eutropha, Alcaligenes eutrophus), optionally autotrophic pseudomonads such as P. aeruginosa, P. saccharophila, P. facilis, P. hydrogenovora, P. hydrogenothermophila, P. carboxydi- hydrogena, P Compransoris, P. carboxydovarans, P. gasotropha and P. stanieri; Rhodopseudomonas palustris, R.
  • Clostridia capable of accepting hydrogen and homoacetate fermentation such as C. aceticum, C. magnum, C. thermoaceticum, C. scatologenes, C. deformicoaceticum and C. thermoautotrophicum; Acetobacterium woodii; Acetogenium kivui; Species of Rhodospirillales (C source carbon monoxide) and Rhodocyclus gelatinosus.
  • the invention is based on the technical problem of providing an improved process for the biotechnological production of organic compounds, especially in the form of C2 to C6 bodies, mainly from carbon dioxide as a carbon source with hydrogen as an "energy source” and means for carrying out this process
  • a technical problem also exists in making such a method usable, in particular, in known microorganisms or cell lines established in biotechnological / fermentative processes, which can be transfected with little effort using conventional recombination technologies to allow a stable synthesis of organic compounds such as carboxylic acids, short-chain fatty acids and alcohol mainly from CO2 as a carbon source and with hydrogen as an "energy source” with high yield, both in anaerobic as well may also occur in aerobic processes.
  • transgenic biological cell in particular a recombinant microorganism or parts thereof, which contains an enzyme equipment which is selected from:
  • cytoplasmic preferably NAD-reducing hydrogenase activity, and / or
  • Enzyme activity suitable for realizing a CO2-fixing metabolic pathway, selected from:
  • RTCC reductive tricarboxylic acid cycle
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • a cell according to the invention especially in the genome, especially in an expression cassette and operably linked to a promoter, contains at least one nucleic acid molecule with a nucleotide sequence which codes for at least one of the aforementioned enzyme activities.
  • the invention also relates to an expression cassette and a vector containing this expression cassette, which are suitable for mediating the expression of the enzyme activities provided according to the invention in the host cell.
  • the expression cassette for transformation of a host cell contains:
  • At least one heterologous nucleic acid molecule encoding enzyme activity selected from: Formyl-tetrahydrofolate ligase activity;
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the invention preferably further provides that the cell additionally contains:
  • the Calvin-Benson-Bassham cycle can additionally be realized for realizing the CO2 fixation, specifically by expressing at least one heterologous enzyme activity selected from:
  • transgenic cell containing at least one recombinant nucleic acid molecule having a nucleotide sequence encoding at least one enzyme activity selected from:
  • Hydrogenase activity preferably cytoplasmic and / or membrane-bound hydrogenase activity
  • Enzyme activity suitable for realizing a CO2-fixing metabolic pathway, selected from:
  • RTCC Reductive tricarboxylic acid cycle
  • an expression cassette according to the invention for transforming a host cell additionally contains: phosphoribulosis kinase activity and ribulose bisphosphate carboxylase activity, nucleotide sequences being present in the expression cassette, in a transformation vector containing the expression cassette and in the host cell to be transformed coding for enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate aldolase activity, absent or absent or inhibiting this enzyme activity.
  • SC serine cycle serine cycle
  • RTCC reductive tricarboxylic acid cycle
  • the invention also provides a process for the biotechnological production of C2-C6 bodies as product of carbon dioxide as substrate and assimilation of hydrogen, use of transgenic biological cell for the biotechnological production of lactate or lactic acid and alcohol from carbon dioxide.
  • the invention makes use of the surprising finding that in a suitable transgenic or recombinant biological cell, in particular by recombinant expression of the abovementioned enzymes or enzyme activities, the fixation of carbon dioxide for the production of organic carbon compounds using an electron donor, especially hydrogen, with high Yield is made possible.
  • the invention thus provides means for carrying out biotechnological processes for a novel, highly effective conversion of, in particular, hydrogen and CO.sub.2 into readily transportable and usable fuels, for example alcohols, as well as basic chemicals and valuable substances, for example carboxylic acids and short-chain fatty acids
  • CO2 as a carbon source thus not only enables the realization of CO2-neutral processes, but also allows an independent choice of location due to the ubiquitous availability of CO2.
  • the present invention Above all, the use of hydrogen as an electron donor as an energy source for microbial processes is exploited.
  • the hydrogen is usually prepared from electrochemical processes from water or from the fermentation of organic waste.
  • the cell expresses at least one of the enzyme activities defined above.
  • the cell expresses two, more, and most preferably all of the enzyme activities defined above.
  • the at least one nucleic acid molecule is operatively linked to an expression system, that is to say with a promoter or promoter system. It is understood that preferably a constitutive promoter is used.
  • the wild type is preferably transformed with a suitable expression vector containing the expression cassette.
  • the transformation of the cells according to the invention and the incorporation of the isolated nucleic acid molecules takes place in a manner known per se, preferably by suitably suitable expression vectors or in conjunction with appropriate expression cassettes, preferably including appropriate expression vectors.
  • the de novo synthesis of nucleic acid molecules takes place with the desired nucleotide sequences, optionally the complete expression cassette in a suitable synthesis system.
  • Transgenic biological cell containing, preferably in the genome and in particular functionally linked to a promoter: (1) at least one nucleic acid molecule encoding hydrogenase activity; and
  • Citrate lyase activity Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the transgenic biological cell contains, preferably in the genome and in particular functionally linked to a promoter, optionally in addition: phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity, wherein enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate- Aldolase activity, not expressed or inhibited.
  • this cell contains, in particular for increasing the synthesis potential, additionally at least one heterologous nucleic acid molecule coding for enzyme activity, selected from:
  • the serine cycle is realized in the cell, preferably in place of the CBB.
  • the cell contains:
  • the cell further has the enzyme activity of a glycine cleavage system.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the cell has the enzyme activity selected from:
  • this cell additionally contains formate dehydrogenase activity for realization using the serine cycle.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the reductive tricarboxylic acid cycle is realized. The cell contains:
  • nucleic acid molecules encoding all of these activities are present, wherein at least one nucleic acid molecule encodes a heterologous enzyme activity.
  • the cell contains: at least one nucleic acid molecule encoding hydrogenase activity selected from:
  • membrane-bound hydrogenase activity preferably from the microorganism E. coli, selected from hydrogenase hvaABC and hydrogenase hybOCAB;
  • cytoplasmic NAD-reducing hydrogenase activity preferably from the microorganism Ralstonia eutropha with at least the structural genes hoxFUYH.
  • the invention basically envisages in all variants that only those genes are recombined heterologously which encode such enzyme activities of a metabolic pathway identified according to the invention. which do not belong to the enzyme equipment of the wild type of the recombined cell.
  • the homologous expression is preferably modified for homologous overexpression.
  • at least one of the enzyme activities involved is expressed both homologously and heterologously.
  • this cell expresses or has a lactate dehydrogenase activity.
  • this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
  • the invention preferably provides that in the cell according to the invention the intermediates formed during the CO2 assimilation can be converted predominantly or exclusively via the activity of a lactate dehydrogenase to lactate.
  • the expression of at least one lactate transporter is additionally realized in the cell according to the invention.
  • the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of a lactate oxidoreductase to lactate.
  • the invention provides that the intermediates formed in the course of CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the methylglyoxal route to lactate.
  • the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of an aldehyde dehydrogenase to lactate.
  • an aldehyde dehydrogenase for enantiomerically pure lactate, the expression of activities of an enantiomer-selective enzyme is realized in the cell according to the invention.
  • the activity of optionally present homologous enzymes with lacking or reverse stereospecificity and, if present, lactate racemase activity is preferably inhibited in the cell according to the invention or preferably eliminated by de-ling of the genes involved.
  • the invention preferably includes those transgenic or recombinant cells selected or derived from bacteria, cyanobacteria, fungi and yeasts.
  • the cell is preferably selected from bacteria of the genera Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Lactobacillus and Lactococcus.
  • the cell is particularly preferably selected from bacteria of the species Escherichia coli, Corynebacterium glutamicum, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum and Clostridium acetobutylicum.
  • the invention also includes those biological cells which are or derived from a synthetic microorganism.
  • a cell is also understood to mean membrane vesicles, membrane particles and parts and fragments thereof.
  • the enzyme activities are preferably located in the "inner” or cytosol or cytoplasm of the cell.
  • at least one of the enzyme activities is associated with a membrane, especially on a cell membrane.
  • the invention accordingly also comprises or parts or fragments of a transgenic invention Microorganism, which are preferred and in a conventional manner and while maintaining the enzyme activity associated or bound to carrier structures.
  • the invention also includes as a "cell" a biocatalyst in which the enzyme activities are realized by isolated or synthesized enzyme proteins having the amino acid sequences given herein, preferably by bypassing cellular expression and translation systems by incorporation of the enzyme proteins into the cell or biocatalyst.
  • Recombinant cells particularly recombinant microorganisms, which contain and express the genes according to the invention are produced in a manner known per se by customary recombination technologies. It is understood that the cells or microorganisms selected for recombination have sufficient resistance to the desired metabolic end products. Preference is given to microorganisms for which standardized cultivation conditions have been established in biotechnological production, so that elaborate adaptation of established biotechnological process management can be minimized or largely avoided.
  • the scope of the invention also covers those cells or microorganisms and cell lines which, even in the non-recombined / transgenic form, contain and express at least one or more of the genes coding for one of the enzyme activities provided according to the invention. It is understood that, depending on the activity of the homologous gene present, it may not be necessary to recombine with the analogous heterologous genes.
  • measures known per se for ensuring the homologous expression or overexpression of the homologous gene are made in order to completely eliminate the metabolic pathway according to the invention. Depending on the enzyme equipment of the starting organism, it may additionally be necessary to eliminate or suppress any existing competing metabolic pathways. This can be done by measures known per se.
  • a preferred embodiment of the invention is a transgenic or recombinant E. coli cell, for example derived from the cell lines K12.
  • An alternative preferred embodiment of the invention is a transgenic or recombinant Ralstonia eutropha cell.
  • nucleic acid molecules can be isolated therefrom and amplified by conventional methods.
  • Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
  • Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle.
  • Figures 3 and 4 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase.
  • Figures 5 and 6 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by a formyl tetrahydrofolate ligase and the key enzymes of the serine cycle.
  • FIGS. 7 and 8 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli, recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase).
  • FIG. 9 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 1. All rivers are hydrogen (100%) based on the intake flow.
  • FIG. 10 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 11 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 3. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 12 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 4. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 13 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 5. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 14 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 6. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 15 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 7. All rivers are based on the uptake of hydrogen (100%).
  • FIG. 16 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. 8. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
  • Hydrogenase activity is understood herein to mean the ability to assimilate elemental hydrogen to form reduction equivalents.
  • the invention thus preferably provides that the host cell is equipped with at least one heterologous hydrogenase operon.
  • at least one homologous hydrogenase activity existing in the WiId type of the cell is additionally expressed.
  • the introduction of the pHG1 megaplasmid from Ralstonia eutropha is preferably provided.
  • the nucleotide sequence of the pHG1 megaplasmid is described in Schwartz et al. 2003 (Schwartz E., Hen A., Cramm R, Eitinger T., Friedrich B., Gottschalk G. (2003).) Complete Nucleotide Sequence of PHG1: A Ralstonia Eutropha H16 Megaplasmid Encoding Key Enzymes of H2-based Lithoautotrophy and Anaerobiosis. J Mol Biol 332, 369-383.) And under Genbank Accession no.
  • the megaplasmid is 452 kbp in size and carries 429 potential genes. Including at least 41 genes for hydrogenase activity. In a preferred variant, it is therefore provided that a shortened or modified megaplasmide is incorporated into the cell according to the invention, which contains at least one hydrogenase operon of the gapase.
  • the invention preferably provides for the recombinant expression of the cytoplasmic NAD-reducing hydrogenase activity from Ralstonia eutropha.
  • the expression of the structural genes hoxFUYH preferably together with the genes involved in the maturation of the enzyme, hypC1, hypD1, hypE1 and hypABF is preferably realized in the cell according to the invention.
  • the expression of the H2 sensor system hoxA, hoxBC, hoxJ is preferred.
  • the heterologous hydrogenase activity preferably originates from the microorganism Ralstonia eutropha, in particular the enzyme NAD hydrogenase (EC 1.12.7.2), or is derived therefrom.
  • the invention makes use of the finding that, above all, the recombinant hydrogenase from Ralstonia eutropha, advantageously comparatively aerotolerant and already uses even the universally usable electron acceptor NAD.
  • the NAD hydrogenase (EC 1.12.7.2) from Ralstonia eutropha is a heterotetramer of 4 subunits.
  • Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 1 for hoxF subunit, SEQ ID NO: 3 for hoxU subunit, SEQ ID NO: 5 for hoxY subunit, and SEQ ID NO: 7 for hoxH subunit.
  • the enzyme protein thus preferably has the amino acid sequences SEQ ID NO: 2, 4, 6, and 8.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, at least three or preferably all heterologous nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1, 3, 5, 7;
  • nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 2, 4, 6, 8;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a hydrogenase activity.
  • the invention preferably further provides for the expression of the hydrogenase activity: the expression of at least one protein involved in the maturation of the hydrogenase activity.
  • the structural genes are selected from: nucleotide sequences encoding SEQ ID NO: 9 for hypC1.
  • the structural genes are selected from: nucleotide sequences encoding SEQ ID NO: 9 for hypC1.
  • the proteins hypA, hypB and hvpF preferably the coding nucleotide sequences SEQ ID NO: 15 for hypA1, SEQ ID NO: 17 for hvpB1 and SEQ ID NO: 19 for hypF1 provided.
  • SEQ ID NO: 21 for hypA2 SEQ ID NO: 23 for hypB2, and SEQ ID NO: 25 for hypF2.
  • the proteins relevant for the maturation thus preferably have the corresponding amino acid sequences SEQ ID NO: 10, 12, 14, as well as 16, 18, 20 and / or 22, 24, 26.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, three, four, five, six, seven, eight or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25;
  • nucleic acid molecules which are suitable for amino acid molecules which have the sequences SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24,
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a maturation protein for hydrogenase activity.
  • the invention preferably further provides in connection with the expression of the hydrogenase activity: the expression of at least one of the proteins involved in the H2 sensor system.
  • the H2 sensor protein is selected from: coding nucleotide sequences SEQ ID NO: 27 for hoxA, SEQ ID NO: 29 for hoxB subunit and SEQ ID NO: 31 for hoxC subunit of hoxBC and SEQ ID NO: 33 for hoxJ ,
  • the H2 sensor proteins therefore preferably have the corresponding amino acid sequences SEQ ID NO: 28, 30, 32 and 34.
  • the H2 sensor system (hoxA, hoxBC, hoxJ) is preferably only realized if a Ralstonia own transcription system is used.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 27, 29, 31, 33;
  • nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 28, 30, 32, 34;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an H 2 sensor Encode protein.
  • the invention provides alternatively or preferably additionally the expression of the activity of a membrane-bound hydrogenase from E. coli.
  • This is especially the enzyme hydrogenase hyaABC and the enzyme hydrogenase hvbOCAB.
  • the invention provides that, alternatively or preferably in addition, the activity of a membrane-bound hydrogenase is expressed.
  • the hydrogenase activity is derived from the microorganism E. coli, especially the enzyme hydrogenase hyaABC or hydrogenase hvbOCAB, or it is derived therefrom.
  • the expression of the membrane-bound hydrogenase hvaABC is realized.
  • the hydrogenase hvaABC from E. coli is a heteromer of 3 subunits. These subunit-encoding nucleotide sequences are SEQ ID NO: 137 for hvaA subunit, SEQ ID NO: 139 for hyaB subunit, and SEQ ID NO: 141 for hyaC subunit.
  • the enzyme protein hydrogenase hvaABC thus, it preferably has the amino acid sequences SEQ ID NO: 138, 140 and 142.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 137, 139, 141;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 138, 140, 142;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
  • the expression of the membrane-bound hydrogenase hybOCAB is realized.
  • the hydrogenase hybOCAB from E. coli is a heteromer of 4 subunits. Nucleotide sequences encoding these subunits are SEQ ID NO: 149 for subunit hybO, SEQ ID NO: 155 for subunit hybC, and SEQ ID NO: 151 for subunit hybA and SEQ ID NO: 153 for subunit hybB.
  • the enzyme protein hydrogenase hybOCAB thus preferably has the amino acid sequences SEQ ID NO: 150, 156, 152 and 154.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which have the sequences SEQ ID NO:
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 150, 152, 154, 156;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
  • the expression of the associated chaperones hyaD, hyaE and hyaF or hybD, hybE, hybF and hybG is also at least partially realized.
  • the nucleotide sequences encoding the chaperones are SEQ ID NO: 143 for hyaD, SEQ ID NO: 145 for hyaE, and SEQ ID NO: 147 for hyaF.
  • the chaperones thus preferably have the amino acid sequences SEQ ID NO: 144, 146 and 148.
  • the chaperone genes hyaE and hyaF are optional and need not be expressed.
  • chaperones hybD, hybE. HybF and hybG coding nucleotide sequences are SEQ ID NO: 157 for hybP, SEQ IP NO: 159 ITy 1 b_E, SEQ IP NO: 161 for hybF and SEQ IP NO: 163 for hybG.
  • chaperones preferably have the amino acid sequences SEQ ID NO: 158, 160, 162 and 164.
  • the invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 143, 145, 147, 157, 159, 161, 163;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 144, 146, 148, 158, 160, 162, 164;
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably code for hydrogenase chaperones.
  • enzymatic activity of an enzyme involved in CBB herein is meant the ability to react an intermediate of the CBB, Calvin cycle or reductive pentose phosphate pathway as a substrate into another intermediate product of the cycle as a product of the enzyme reaction.
  • 11 enzymes are involved in CBB.
  • the CBB can be divided into three sections: actual carbon fixation (carboxylation) tion), reduction phase (a C3 body is released as product per three cycles) and regeneration phase of the carbon acceptor ribulose-1, 5-bisphosphate.
  • the activities of a phosphoribulose kinase (phosphoribulokinase) and a ribulose bisphosphate carboxylase are considered as key enzyme activities of CBB.
  • the invention provides for the realization of the CBB in the cell according to the invention: the optionally heterologous expression of at least one phosphoribulose kinase activity and one ribulose bisphosphate carboxylase activity.
  • Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate in Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
  • the invention provides, above all, that in the cell according to the invention the expression of fructose bisphosphatase activity and / or of fructose bisphosphate aldolase activity by deletion of these Activity-encoding nucleotide sequences is switched off.
  • the invention provides, above all, for predominantly or exclusively the Calvin-Benson-Bassham cycle (CBB) to be implemented for CO2 assimilation, wherein at least one enzyme activity involved in CBB is sufficiently expressed in the cell according to the invention. It is preferably provided that the expression of activities of enzymes selected from: Phosphoribulose kinase activity (phosphoribulokinase) and ribulose bisphosphate carboxylase activity
  • the invention further provides that the Calvin-Benson-Bassham cycle (CBB), while avoiding, inhibiting or deleting at least one, preferably both, enzyme activity optionally present in the wild type of the cell, is selected from:
  • the invention provides that the CBB is realized by inhibiting or deleting a fructose-bisphosphatase activity. This is preferably done according to the invention by inhibiting the expression and / or deletion of at least one nucleotide sequence coding for this enzyme activity or at least one of the genes. In connection with the use of a recombinant E. coli cell, the invention preferably provides for this:
  • the inhibition of the activity of the fructose bisphosphatase I fbp_ preferably by deletion of at least the gene for fbj), represented by the SEQ ID NO: 77 or coding for the amide Noklaresequenz SEQ ID NO: 78, and alternatively or preferably in addition
  • the inhibition of the activity of the fructose bisphosphatase II glpX preferably by deletion of at least the gene for glpX, represented by the SEQ ID NO: 79 or coding for the
  • the invention provides that the CBB is realized by inhibiting or deleting a fructose bisphosphate aldolase activity. This is done according to the invention preferably by inhibiting the expression and / or deletion of at least one nucleotide sequence encoding this enzyme activity or at least one of the genes.
  • the invention preferably additionally or alternatively additionally provides for this:
  • fructose bisphosphate aldolase II activity fbaA by deleting at least the gene for fbaA represented by SEQ ID NO: 81 or coding for the amino acid sequence SEQ ID NO: 82, and alternatively or preferably additionally
  • fructose bisphosphate aldolase I activity fbaB by deletion of at least the gene for fbaB represented by SEQ ID NO: 83 or coding for the amino acid sequence SEQ ID NO: 84.
  • the phosphoribulose kinase activity is from the microorganism Ralstonia eutropha, especially the enzyme Phosphoribulose kinase cbbPp, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 35.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 36.
  • the ribulose bisphosphate carboxylase activity is derived from the microorganism Ralstonia eutropha, especially the enzyme ribulose bisphosphate carboxylase cbbSp / cbbLp, or is derived therefrom.
  • the ribulose bisphosphate carboxylase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 37 for subunit cbbLp and SEQ ID NO: 39 for subunit cbbSp.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 40 and 42.
  • the invention preferably provides for the realization of the CBB in the cell according to the invention in the cell, the pHG1 megaplasmid from Ralstonia eutropha is present and there, in addition to the hydrogenase operon especially the phosphoribulose kinase operon and / or the Ribulose bisphosphate carboxylase operon are expressed. It is provided according to the invention to use a truncated or modified pHG1 megaplasmid, wherein the operons coding for fructose bisphosphatase activity and fructose bisphosphate aldolase activity are deleted or suppressed.
  • the invention preferably relates to a cell which contains at least one, preferably at least two or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 35, 37, 39;
  • nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 36, 38, 40;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a phosphoribulose kinase activity and / or encode a ribulose bisphosphate carboxylase activity.
  • the expression of a fructose-6-phosphate aldolase activity is realized.
  • the fructose-6-phosphate aldolase activity is derived from the microorganism E. coli, especially the enzyme fructose-6-phosphate aldolase fsaA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 85.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 86.
  • the enzyme is fructose 6-phosphate aldolase fsaB, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 87.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 88.
  • the expression of a glycerol-3-phosphate dehydrogenase activity is realized.
  • the glycerol-3-phosphate dehydrogenase Activity from the microorganism E. coli, especially the enzyme glycerol-3-phosphate dehydrogenase gpsA, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 89.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 90.
  • the enzyme glycerol-3-phosphate dehydrogenase qlpABC is preferred, or it is derived therefrom. According to the current state of knowledge, this glycerol-3-phosphate dehydrogenase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 91 for subunit qlpA, SEQ ID NO: 93 for subunit qlpB and SEQ ID NO: 95 for subunit qlpC.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 92, 94 and 96.
  • the enzyme glycerol-3-phosphate dehydrogenase qlpD is preferred, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 97.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 98.
  • the expression of a glycerol kinase activity is realized.
  • the glycerol kinase activity is derived from the microorganism E. coli, especially the enzyme glycerol kinase glpK, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 99.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 100.
  • the expression of a glycerol dehydrogenase activity is realized.
  • the glycerol dehydrogenase activity comes from the microorganism E. coli, especially the enzyme glycerol dehydrogenase gldA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 101.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 102.
  • transaldolase activity preferably originates from the microorganism E. coli, in particular the enzyme is the transaldolase talA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 103.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 104.
  • the enzyme is the transaldolase talB, or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 105.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 106.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105;
  • nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 include or consist of encode;
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an enzyme activity selected from:
  • Fructose-6-phosphate aldolase activity Fructose-6-phosphate aldolase activity, glycerol-3-phosphate dehydrogenase activity, glycerol kinase activity,
  • Glycerol dehydrogenase activity and transaldolase activity are Glycerol dehydrogenase activity and transaldolase activity.
  • Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle; Fructose bisphosphate aldolase and fructose bisphosphatase have been replaced by glycerol-3-phosphate dehydrogenase, glycerol kinase, glycerol dehydrogenase, fructose-6-phosphate aldolase and transaldolase to increase their synthetic potential.
  • the serine cycle (SC) is also available according to the invention. Due to the high
  • the energy requirement of fixing CO2 in CBB is a production of organic substances with this path of CO2 fixation (eg lactate) only possible if, in addition to CO2, another electron acceptor, for example oxygen, is available.
  • CO2 fixation eg lactate
  • the ribulose bisphosphate carboxylase preferably used in CBB has a comparatively low activity.
  • SC there is advantageously the possibility in principle to completely dispense with an additional electron acceptor besides CO2.
  • the SC is not dependent on the fixation of CO2 in the reaction of ribulose bisphosphate carboxylase.
  • this enzyme also catalyzes an undesirable side reaction with O 2 as a substrate, which reduces the efficiency of the metabolic pathway.
  • the invention preferably provides in this alternative variant for predominantly or exclusively the serine cycle (SC) to be implemented for CO2 assimilation.
  • SC serine cycle
  • the realization of this metabolic pathway according to the invention primarily provides for the expression of a formyl-tetrahydrofolate ligase activity.
  • Fig. 3 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by a formyl tetrahydrofolate ligase; For the assimilation of hydrogen and for the synthesis of glycine, homologous enzymes are used.
  • Fig. 4 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase; for the synthesis of glycine, homologous enzymes are used; the assimilation of hydrogen is by a recombinant hydrogenase.
  • the realization according to the invention of a formyl-tetrahydrofolate ligase activity ensures, above all, the introduction of formate into the transfer of C 1 -substances, in particular the transfer of C 1 -substances to glycine in connection with a serine hydroxymethyltransferase activity. This process is particularly relevant in connection with the use of E. coli as a host cell.
  • the homologous or heterologous expression of a, preferably cytosolic, formate dehydrogenase activity and / or a comparable activity is realized.
  • At least one formyl-tetrahydrofolate ligase activity is expressed according to the invention.
  • the formyl tetrahydrofolate ligase activity preferably originates from the microorganism Methylobacterium extorquens, especially the enzyme formyl tetrahydrofolate ligase ftfL, or is derived from it.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 107.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 108.
  • the invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequence SEQ ID NO: 107;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of formyl tetrahydrofolate ligase encode.
  • the SC is realized by including a glycine cleavage system activity present in the host cell.
  • a glycine cleavage system activity is expressed.
  • the glycine cleavage system activity qcvPHT and Issd is from E. coli or derived therefrom.
  • the glycine cleavage system of E. coli is composed of 4 subunits.
  • Preferred, subunit-encoding nucleotide sequences are SEQ ID NO: 189 for subunit qcvP.
  • the enzyme system thus preferably has the amino acid sequences SEQ ID NO: 190, 192, 194 and 64.
  • the invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 189, 191, 193, 63;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 190, 192, 194, 64;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of the glycine cleavage Encode system.
  • the recombinant expression of activities of key enzymes of the SC selected from:
  • an enzyme activity of an enzyme involved in the SC herein is meant the ability to react an intermediate of the SC as a substrate into another intermediate of the cycle as a product of the enzyme reaction.
  • key enzymes of the SC are understood as meaning serine-glyoxylate aminotransferase, hydroxypyruvate reductase, malate thiokinase, glycerol dehydrogenase MaIyI-CoA lyase and isocitrate lyase.
  • all of the aforementioned enzyme activities are expressed, preferably all enzyme activities involved in SC are expressed to a sufficient extent.
  • at least one, more preferably at least two of these enzyme activities are heterologously expressed.
  • this allows circumvention of the glycine cleavage system activity present in connection with the use of E. coli as the host cell. It also uses the amino group from serine to synthesize glycine from glyoxylate.
  • the realization of a hydroxypyruvate reductase activity which is preferred according to the invention permits the conversion of the metabolic product of the serine-glyoxylate aminotransferase together with the glycerate kinase in the Embden-Meyerhof-Pamas route, which is preferably realized in the cell according to the invention.
  • the regeneration of glyoxylate is optional, alternatively or preferably in addition, via a homologous phosphoenolpyrovate carboxylase activity, tricarboxylic acid and glyoxylate pathway; This is especially relevant in connection with the use of E. coli as the host cell.
  • the preferred realization of the isocitrate-lyase activity according to the invention is preferably also active as part of the glyoxylate pathway at the glyoxylate regeneration in the serine pathway. This is particularly relevant in connection with the use of E. coli as a host cell.
  • the serine-glyoxylate aminotransferase activity derives from the microorganism Methylobacterium extorquens, especially the enzyme serine-glyoxylate aminotransferase sgaA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 113.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 114.
  • the hydroxypyruvate reductase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme hydroxypyruvate reductase is hprA or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 1 15.
  • the enzyme protein therefore preferably has the amino acid sequence SEQ ID NO: 116.
  • the serine malate thiokinase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme malate thiokinase is mtkAB, or is derived therefrom.
  • the malate thiokinase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 117 for mtkA subunit and SEQ ID NO: 119 for mtkB subunit.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 118 and 120.
  • the glycerol dehydrogenase malyl CoA lyase activity is derived from the microorganism Methylobacterium extorquens, especially the enzyme glycerol dehydrogenase malyl CoA lyase mcIA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 121.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 122.
  • the isocitrate lyase activity preferably originates from E. coli, in particular the enzyme isocitrate lyase aceA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 11.1.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 1 12.
  • expression of isocitrate lyase activity is realized by homologous expression, preferably by homologous overexpression.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five or preferably all nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1 1 1, 1 13, 1 15, 1 17, 1 19;
  • nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 1 12, 1 14, 1 16, 1 18, 120;
  • nucleic acid molecules which, with the nucleic acid molecules described under a) and b), are at least 50%, preferably at least 60%, 70%, 80%, particularly preferably have at least 90% or more homology and prefer the activity of key enzymes of the SC selected from: serine-glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, malate-thiokinase activity, glycerol dehydrogenase malyl-CoA-lyase-
  • the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB ,
  • the genes for phosphoribulose kinase cbbPp SEQ ID NO: 35
  • ribulose bisphosphate carboxylase cbbLp SEQ ID NO: 37
  • cbbSp SEQ ID NO: 39.
  • the invention also provides the reductive tricarboxylic acid cycle (RTCC).
  • RTCC reductive tricarboxylic acid cycle
  • enzyme activity of an enzyme involved in the RTCC herein is meant the ability to react an intermediate of the RTCC as a substrate into another intermediate of the cycle as a product of the enzyme reaction.
  • Key enzymes of the RTCC are understood to mean citrate lyase, oxoglutarate oxidoreductase and fumarate reductase.
  • the invention preferably provides for predominantly or exclusively the reductive tricarboxylic acid cycle (RTCC) to be carried out for CO2 assimilation, wherein all the enzyme activities involved in the RTCC are sufficiently expressed in the cell according to the invention.
  • RTCC reductive tricarboxylic acid cycle
  • the expression of activities of key enzymes of the RTCC selected from:
  • Citrate lyase activity oxoglutarate oxidoreductase activity
  • Fumarate reductase activity and / or comparable activities are realized.
  • all of the abovementioned enzyme activities are expressed, preferably all enzyme activities involved in the RTCC are expressed to a sufficient extent.
  • at least one, more preferably at least two, of these enzyme activities are heterologously expressed.
  • the citrate-lyase activity preferably originates from the microorganism Chlorobium tepidum, especially the enzyme citrate-lyase ac-IAB, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 123 for subunit acIA, and SEQ ID NO: 125 for subunit acIB. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 124 and 126.
  • the citrate lyase activity is from E. coli, especially the enzyme citrate lyase citFED, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 127 for subunit citF, SEQ ID NO: 129 for subunit citE, and SEQ ID NO: 131 for subunit citD.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 128, 130 and 132.
  • the expression of an active citrate lyase activity is realized by homologous expression, preferably by homologous overexpression of, possibly by spontaneous mutation, mutant allele of the originally inactive citrate lyase of E. coli .
  • the oxoglutarate oxidoreductase activity preferably originates from the microorganism Hydrogenobacter thermophilus, in particular the enzyme oxoglutarate oxidoreductase korAB, or is derived therefrom. According to the current state of knowledge, this oxoglutarate oxidoreductase is composed of 2 subunits.
  • Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 133 for subunit korA, and SEQ ID NO: 135 for subunit korB.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 134 and 136.
  • the fumarate reductase activity is from E. coli, especially the enzyme fumarate reductase is frdDCBA or derived therefrom.
  • the fumarate reductase is composed of 4 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 57 for subunit frdD, SEQ ID NO: 55 for subunit frdC, SEQ ID NO: 53 for subunit frdB and SEQ ID NO: 51 for subunit frdA.
  • the enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 58, 56, 54 and 52.
  • the expression of fumarate reductase activity is realized by homologous expression, preferably by homologous overexpression.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain the sequences SEQ ID NO: 51, 53, 57, 123, 125, 127, 129, 131, 133, 135 or consist thereof;
  • nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 52, 54, 56, 58, 124, 126, 128,
  • nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably an activity of key enzymes of the RTCC selected from citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
  • the homologous and preferably the heterologous expression of a, preferably reversible, pyruvate: ferredoxin oxidoreductase activity or pyruvate synthase activity and / or an activity comparable therewith is realized.
  • a, preferably reversible, pyruvate: ferredoxin oxidoreductase activity or pyruvate synthase activity and / or an activity comparable therewith is realized.
  • E. coli or Pseudomonas putida as the host cell is provided in an alternative variant, that preferably by the homologous or heterologous expression of enzyme activity of the malate pathway, preferably by an NAD-dependent malate enzyme activity, the NAD Dependent malate enzyme from E. coli, the intermediate oxaloacetate is irreversibly converted into pyruvate by decarboxylation.
  • the intermediate oxaloacetate by homologous or optionally heterologous expression of oxaloacetate decarboxylase activity, preferably the E. coli oxaloacetate decarboxylase.
  • oxaloacetate decarboxylase activity preferably the E. coli oxaloacetate decarboxylase.
  • the homologous overexpression of at least one of the aforementioned enzyme activities is provided to advantageously prevent byproducts from the tricarboxylic acid cycle from being produced instead of lactate.
  • this measure is preferably provided in order to realize the irreversible outflow from the tricarboxylic acid cycle.
  • this is usually associated with a pyruvate: ferredoxin oxidoreductase activity.
  • Figure 8 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase).
  • oxygen is used as the terminal electron acceptor.
  • a recombi- nant hydrogenase is used for the assimilation of hydrogen.
  • the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB ,
  • the genes for phosphoribulose kinase cbbPp and / or ribulose bisphosphate carboxylase cbbLp and cbbSp are preferably deleted on the megaplasmid.
  • the invention preferably further contemplates suppressing the potential degradation of lactate precursors and directing the material flow toward lactate. This is preferably realized by inhibiting any enzyme activities of competing metabolic pathways and / or comparable enzyme activities present in the cell according to the invention, preferably by inhibiting the expression and / or deletion of the nucleotide sequences or genes coding for these enzyme activities.
  • the production of lactic acid further provided to suppress acetate-converting pathways to suppress the formation of acetate as an undesired by-product.
  • the CBB is preferably at least one enzyme, preferably all enzymes selected from:
  • Acetate Kinase A / Propionate Kinase 2 Activity Phosphate Acetyl Transferase Activity, Phosphoenolpyruvate Carboxylase Activity, Pyruvate Formate Lyase I Activity, Acetaldehyde CoA Dehydrogenase Activity,
  • Fumarate reductase activity pyruvate dehydrogenase activity, succinate dehydrogenase activity
  • At least one enzyme preferably all enzymes, selected from:
  • the invention preferably provides for this: the inhibition of the phosphate acetyltransferase activity preferably by deletion of at least one of the genes for p_ta, represented by the SEQ ID NO: 41 or coding for the amino acid sequence SEQ ID NO: 42, and alternatively or preferably in addition
  • CBB optionally (in CBB) the inhibition of the phosphoenolpyruvate carboxylase activity, preferably by deletion of at least one of the genes for ppc, represented by SEQ ID NO: 43 or coding for the amino acid sequence SEQ ID NO: 44, wherein the inhibition of phosphoenolpyruvate
  • Carboxylase activity ppc is less preferred in this context.
  • the invention provides alternatively or preferably additionally:
  • the inhibition of pyruvate formate lyase I activity is preferred by deleting at least one of the genes for pflB, represented by SEQ ID NO: 45 or coding for the amino acid sequence SEQ ID NO: 46, and alternatively or preferably additionally
  • the inhibition of the acetate kinase A / propionate kinase 2 activity is preferred by deleting at least one of the genes for ackA represented by SEQ ID NO: 47 or coding for the amino acid sequence SEQ ID NO: 48, and alternatively or preferably additionally the inhibition of acetaldehyde CoA dehydrogenase activity preferably by deletion of at least one of the genes for ad ⁇ hE represented by SEQ ID NO: 49 or coding for the amino acid sequence SEQ ID NO: 50, and alternatively or preferably additionally
  • CBB optionally (in CBB) the inhibition of anaerobic fumarate reductase activity, preferably by deletion of at least one of the genes for frdABCD, represented by SEQ ID NOS: 51, 53, 55 and 57 or coding for the amino acid sequences SEQ ID NO : 52, 54, 56 and 58, more preferably frdA and / or frdB.
  • the invention alternatively provides for this:
  • the inhibition of pyruvate dehydrogenase activity is preferred by deleting at least one of the aceEF / lpdA genes represented by SEQ ID NOs: 59 and 61, 63 or coding for the amino acid sequences SEQ ID NOs: 60 and 62, and alternatively or preferably additionally
  • Activity is preferred by deleting at least one of the genes for ackA and alternatively or preferably additionally
  • the inhibition of acetaldehyde-CoA dehydrogenase activity is preferred by deleting at least one of the genes for ad; ITE and alternatively or preferably in addition the inhibition of succinate dehydrogenase activity, preferably by deletion of at least one of the genes for sdhABCD represented by SEQ ID NO: 65, 67, 69 and 71 or coding for the amino acid sequence SEQ ID NO: 66, 68, 70 and 72, especially preferably from sdhA and / or sdhB.
  • the invention preferably provides that the expression of a D-lactate dehydrogenase activity and / or a comparable activity is realized.
  • the D-lactate dehydrogenase activity originates from E. coli, in particular it is the enzyme lactate dehydrogenase IdhA, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 73.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 74.
  • the D-lactate dehydrogenase activity originates from Lactobacillus plantarum, in particular it is the enzyme lactate dehydrogenase IdhD, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 185.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 186.
  • the expression of D-lactate dehydrogenase activity is realized by homologous expression, preferably by homologous overexpression.
  • homologous expression In the context of an aerobic process, heterologous expression or homologous overexpression of lactate dehydrogenase activity is mandatory.
  • the D-lactate transporter activity preferably originates from E. coli, in particular it is the transporter HdP or IctP, or is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 165.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 166.
  • the invention thus preferably relates to a cell which contains at least one, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 73, 165, 185;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 74, 166, 186;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a D-lactate dehydrogenase activity and / or D-lactate transporter activity.
  • the invention additionally provides: the inhibition of homologous L-lactate dehydrogenase activity, preferably by deletion of the genes for HdD represented by SEQ ID NO: 75 or coding for the amino acid sequence SEQ ID NO: 76.
  • the invention additionally provides:
  • the inhibition of the homologous L-lactate dehydrogenase activity preferably by deletion of the gene for IdhLI, represented by SEQ ID NO: 167 or coding for the amino acid sequence SEQ ID NO: 168, and alternatively and preferably additionally by deletion of the gene for ldhL2 represented by SEQ ID NO: 169 or coding for the amino acid sequence SEQ ID NO: 170, and alternatively or preferably additionally
  • the inhibition of the homologous lactate racemase activity preferably by deletion of at least one of the genes for Ia 1 rABC1 C2E qlpFL represented by SEQ ID NO: 173, 175, 177, 179 and 181 or coding for the amino acid sequence SEQ ID NO: 174, 176, 178, 180 and 182.
  • the invention preferably provides that the expression of an L-lactate dehydrogenase activity and / or a comparable activity is realized.
  • the L-lactate dehydrogenase activity preferably originates from E. coli, in particular it is the enzyme L-lactate: quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD, or is derived therefrom.
  • this activity co- nucleotide sequence is SEQ ID NO: 75.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 76.
  • the homologous expression of L-lactate dehydrogenase activity is preferably realized by homologous overexpression of L-lactate-quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD.
  • the L-lactate dehydrogenase activity originates from the microorganism Lactobacillus plantarum. It is preferably the enzyme lactate dehydrogenase ldhL.1 or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 167.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 168.
  • it is the enzyme lactate dehydrogenase ldhL2 or it is derived therefrom.
  • a preferred nucleotide sequence encoding this activity is SEQ ID NO: 169.
  • the enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 170.
  • the invention thus preferably relates to a cell which contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
  • nucleic acid molecules which have the sequences SEQ ID NO:
  • 167, 171, 75 contain or consist of;
  • nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 168, 172, 76;
  • nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an L-lactate dehydrogenase activity and / or encode an L-lactate transporter activity.
  • the invention additionally provides:
  • the inhibition of the homologous fermentative D-lactate dehydrogenase activity preferably by deletion of the genes for IdhA, represented by SEQ ID NO: 73 or coding for the amino acid sequence SEQ ID NO: 74.
  • the invention additionally provides: the inhibition of the homologous D-lactate dehydrogenase activity, preferably by deletion of the genes for d] d, represented by SEQ ID NO: 187 or coding for the amino acid sequence SEQ ID NO: 188.
  • the invention additionally provides:
  • the inhibition of the homologous D-lactate dehydrogenase activity preferably by deletion of the gene for IdhD, represented by SEQ ID NO: 185 or coding for the amino acid sequence SEQ ID NO: 186, and alternatively or preferably additionally
  • the above-characterized recombinant microorganisms or transgenic biological cells according to the invention are particularly suitable for the biotechnological synthesis of lactate / lactic acid and other carboxylic acids or short-chain fatty acids. These are according to the invention C2 to C6 body, preferably C3 to C6 body.
  • Preferred metabolic end products which can be produced by means of the cells according to the invention are short-chain carboxylic acids, especially monocarboxylic and dicarboxylic acids, preferably D-lactate and L-lactate, acetate / acetic acid, formic acid / formic acid and succinate / succinic acid, and also mono- and dicarboxylic acids polyhydric alcohols, preferably ethanol. They serve as starting materials for further organic synthesis, for example for the production of plastics (eg polylactates).
  • the products which can be prepared according to the invention can also be used as energy carriers, especially as fuels / fuels and for their production.
  • the invention also provides a process for the biotechnological production of organic carbon compounds, especially C2 to C6 bodies, as a product of CO2 as substrate and an inorganic electron donor such as hydrogen, wherein in step (a) a cell according to the invention is provided, in step (b) the cell is contacted with the substrate and the electron donor and in step (c) the cell is cultured under conditions whereby the substrates are reacted and the cell forms the organic carbon bonds.
  • the product is isolated from the culture medium and / or the cell and optionally purified. Usually, the intracellularly formed product is released from the cell into the extracellular medium.
  • the cultivation preferably takes place in preferably liquid culture medium. It is preferably provided to cultivate the cell under anaerobic conditions. Depending on the host organism, it is also provided in an alternative variant to cultivate the cell under aerobic conditions. On the basis of the above description of the invention, the person skilled in the art can choose the respectively favorable enzyme equipment.
  • the electron donor is preferably elementary hydrogen, preferably gaseous hydrogen. Preferred alternative electron donors are selected from hydrogen sulfide, elemental sulfur, sulfite, thiosulfate, ammonium, nitrite and metals in reduced form.
  • At least one compound selected from O 2 is used as an electron acceptor.
  • nitrate, nitrite, sulfate, sulfite, thiosulfate and fumarate is used as an electron acceptor.
  • HypD1 involved in metallocene formation of hydrogenases
  • HypA1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H 16] EC number
  • HoxC Designation HoxC component of the hydrogen sensing and signal transduction system
  • HoxJ histidine protein kinase component of the hydrogen senso ⁇ ng and signal transduction system Abbreviation hoxJ Designation HoxJ histidine protein kinase component of the hydrogen senso ⁇ ng and signal transduction system
  • NCBI GenelD 946778 DNA sequence SEQ ID NO 41 amino acid sequence SEQ ID NO 42 Abbreviation ppc
  • DNA sequence SEQ ID NO 43 amino acid sequence SEQ ID NO 44 Abbreviation pflb Name pyruvate formate lyase I [Escherichia coli
  • E3 component is part of three enzyme complexes, eg of pyruvate dehydrogenase [Escherichia coli K12]
  • NCBI-GenelD 946632 DNA sequence SEQ ID NO 83 amino acid sequence SEQ ID NO 84 Abbreviation fsaA
  • fructose-6-phosphate aldolase 1 [Escherichia coli K12]
  • fructose-6-phosphate aldolase 2 [Escherichia coli K12]
  • DNA sequence SEQ ID NO 91 amino acid sequence SEQ ID NO 92 Abbreviation glpB Name sn-glycerol-3-phosphate dehydrogenase
  • transaldolase A Esscherichia coli K12
  • transaldolase B Esscherichia coli K12
  • NCBI-GI 439605 (detail) NCBI-GenelD DNA sequence SEQ ID NO 113 amino acid sequence SEQ ID NO 114
  • mtkB Methylobacte ⁇ um extorquens malate thiokinase (alpha subunit)
  • citrate lyase, subunit 2 [Chlorobium tepi dum TLS]
  • citrate lyase, subunit 1 [Chlorobium tepidum TLS]
  • citrate lyase citrate-ACP transferase (alpha) subunit [Escherichia coli K12]
  • citrate lyase citryl-ACP lyase (beta) subunit [Escherichia coli K12]
  • citrate lyase acyl car ⁇ er (gamma) subunit [Escherichia coli K12]
  • HyaA and HyaB proteins [Escherichia coli K12]
  • DNA sequence SEQ ID NO 153 amino acid sequence SEQ ID NO 154 Abbreviation hybC Name hydrogenase 2, large subunit [Escherichia coli K12]
  • NCBI GenelD 945182 DNA sequence SEQ ID NO 155 amino acid sequence SEQ ID NO 156 Abbreviation hybD
  • IctP Name L-lactate transport protein [Lactobacillus plantarum WC FS 1]
  • gcvT Name aminomethyltransferase, tetrahydrofolate-dependent, subunit (T protein) of glycine cleavage complex [Escherichia coli K12]
  • Exemplary embodiment Synthesis of lactic acid from CO2 and H2 in a recombinant E. coli strain
  • the starting point for the transformation is E. coli strain K12.
  • expression cassettes were used for: NAL-reducing cytosolic hydrogenase activity from Ralstonia, structural hoxFUYH and maturation genes hvpC1, hypD1, hypE1 and hypABF, as well as formyl-tetrahydrofolate ligase activity from mammothacterium, especially containing nucleic acid molecules having the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 107, in expression vectors pUC or pPCU18 or the like cloned / ligated.
  • the ligation of the plasmid DNA is carried out in a conventional manner.
  • the vector is opened by hydrolysis with restriction endonuclease and the corresponding DNA sequences are inserted. Ligation takes place, for example, by cassette mutagenesis.
  • transformation-competent E. coli cells and the production of transformation-competent E. coli cells is preferably carried out according to the protocol of Hanahan, 1985 (Hanahan, D. in Glover, DM (ed.)) DNA Cloning: "A Practical Approach” IRL Press Oxford 109-135).
  • E. coli K12 strains for example TH1, TH2, TH5, TH5 ⁇ , C600, Top 10, XL1-Blue and their derivatives are used.
  • a fresh cell colony is suspended in 5 ml SOB medium and cultured with shaking at 37 ° C to a cell density of 1 to 2 x 10 8 / ml. Subsequently, 1: 1 with 40% glycerol / 60% SOB medium (1% yeast extract, 2% Bacto-trypton-mmol / l NaCl, 2.5 mmol / l KCl, after autoclaving to 10 mmol / l MgCl 2 and MgSO 4 ) diluted and cooled on ice. From the pre-culture, a cell smear is applied to an LM plate and incubated overnight at 37 ° C.
  • the cell suspension may, for example in 1, 5 ml reaction vessels after slow deep-freezing are stored at -70 0 C for several months.
  • the ligation is carried out in a molar ratio of DNA to vector DNA of 1: 1 to 1: 3.
  • the ligation mixture 4 .mu.l of sterile water, 2 .mu.l of 5 ⁇ Ligasebuffer (250 mmol / l Tris-HCl, pH 7.6, 50 mmol / l MgCl 2 , 5 mmol / l ATP, 5 mmol / l DTT, 25% PEE ( w / v)), 1 ml of amplified DNA, 2 ⁇ l of vector DNA (with 3 'T overhang in 1 ⁇ l T4).
  • Ligase is placed in a 1, 5 ml reaction vessel and incubated after mixing overnight at 14 ° C. In each case 1-5 ⁇ l of the mixture are used to transform transformation-competent E. coli cells; where appropriate, the ligation mixture may be stored at -20 0 C.
  • selection plates are used, in each case 100 .mu.l of a mixture of 10 .mu.l 100 mmol / l IPTG, 40 .mu.l 3% X-GaI and 50 .mu.l SOC medium are plated on an LB-AMP agar plate and this for about Incubated for 1 hour at 37 ° C. After incubation, in each case 200 ⁇ l of the cell suspension are plated out on the selection plate. On the basis of the ⁇ -complementation test, the colonies which do not contain a recombinant plasmid can easily be distinguished from the recombinant clones which have no coloration due to the blue coloration. The stable heterologous expression in the so available Recombinant E. coli cells are verified in a conventional manner.
  • the resulting recombinant E. coli cells are prepared after inoculation in a 50 L fermenter with culture medium and cultured at 25 to 40 0 C.
  • the culture is cultured with introduction of gaseous CO2 and H2 in the reactor.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Carbon And Carbon Compounds (AREA)

Abstract

The invention relates to the biotechnological use of inorganic electron donors, above all of gaseous hydrogen for fixing carbon dioxide as a carbon source for synthesising organic compounds as energy carriers and end products, by fermentation in micro-organisms.

Description

Biotechnoloqische Fixierung von Kohlenstoffdioxid Biotechnological fixation of carbon dioxide
Beschreibungdescription
Die Erfindung betrifft die biotechnologische Nutzung von anorgani- sehen Elektronendonatoren, vor allem von gasförmigem Wasserstoff zur Fixierung von Kohlenstoffdioxid als Kohlenstoffquelle zur Synthese von organischen Verbindungen als Energieträger und Wertprodukte durch Fermentation in Mikroorganismen.The invention relates to the biotechnological use of anorgani- see electron donors, especially of gaseous hydrogen for fixing carbon dioxide as a carbon source for the synthesis of organic compounds as energy sources and value products by fermentation in microorganisms.
Die Umsetzung von Wasserstoff und Kohlenstoffdioxid in einem bio- logischen Organismus ist bekannt. Es wurden Wildtypen verschiedener Mikroorganismen entdeckt, die zu dieser Stoffwechselleistung fähig sind. Dazu zählen Ralstonia metallidurans, Ralstonia metallidu- rans (Ralstonia eutropha; Alcaligenes eutrophus), fakultativ au- totrophe Pseudomonaden wie P. aeruginosa, P. saccharophila, P. facilis, P. hydrogenovora, P. hydrogenothermophila, P. carboxydo- hydrogena, P. compransoris, P. carboxydovarans, P. gasotropha und P. stanieri; Rhodopseudomonas palustris, R. capsulata; zur Wasserstoff-Aufnahme und Homoacetatgärung fähige Clostridien wie C. aceticum, C. magnum, C. thermoaceticum, C. scatologenes, C. for- micoaceticum und C. thermoautotrophicum; Acetobacterium woodii; Acetogenium kivui; Arten der Rhodospirillales (C-Quelle Kohlenmo- nooxid) und Rhodocyclus gelatinosus.The reaction of hydrogen and carbon dioxide in a biological organism is known. Wild types of various microorganisms have been discovered that are capable of this metabolic activity. These include Ralstonia metallidurans, Ralstonia metallidurans (Ralstonia eutropha, Alcaligenes eutrophus), optionally autotrophic pseudomonads such as P. aeruginosa, P. saccharophila, P. facilis, P. hydrogenovora, P. hydrogenothermophila, P. carboxydi- hydrogena, P Compransoris, P. carboxydovarans, P. gasotropha and P. stanieri; Rhodopseudomonas palustris, R. capsulata; Clostridia capable of accepting hydrogen and homoacetate fermentation, such as C. aceticum, C. magnum, C. thermoaceticum, C. scatologenes, C. deformicoaceticum and C. thermoautotrophicum; Acetobacterium woodii; Acetogenium kivui; Species of Rhodospirillales (C source carbon monoxide) and Rhodocyclus gelatinosus.
Eine Vielzahl dieser Organismen eignen sich nicht oder nicht ohne weiteres zur Verwendung in der biotechnologischen Anwendung. Darüber hinaus sind vor allem geringe Umsatzraten realisiert. AufgabenstellungMany of these organisms are not or not readily suitable for use in biotechnological applications. In addition, especially low conversion rates are realized. task
Der Erfindung liegt das technische Problem zugrunde, ein verbessertes Verfahren zur biotechnologischen Herstellung von organischen Verbindungen, besonders in Form von C2 bis C6-Körpern vor allem aus Kohlenstoffdioxid als Kohlenstoffquelle unter Umsetzung mit Wasserstoff als „Energiequelle" sowie Mittel zur Durchführung dieses Verfahrens bereitzustellen. Das technische Problem besteht auch darin, ein solches Verfahren besonders auch bei bekannten, in bio- technologischen/fermentativen Prozessen etablierten Mikroorganis- men beziehungsweise Zelllinien, die mittels üblicher Rekombinationstechnologien unter geringem Aufwand transfiziert werden können, einsetzbar zu machen. Gleichzeitig soll das technische Problem gelöst werden, eine stabile Synthese von organischen Verbindungen wie Carbonsäuren, kurzkettige Fettsäuren und Alkohol vor allem aus CO2 als Kohlenstoffquelle und mit Wasserstoff als „Energiequelle" mit hoher Ausbeute zu ermöglichen, die sowohl in anaeroben als gegebenenfalls auch in aeroben Prozessen ablaufen kann.The invention is based on the technical problem of providing an improved process for the biotechnological production of organic compounds, especially in the form of C2 to C6 bodies, mainly from carbon dioxide as a carbon source with hydrogen as an "energy source" and means for carrying out this process A technical problem also exists in making such a method usable, in particular, in known microorganisms or cell lines established in biotechnological / fermentative processes, which can be transfected with little effort using conventional recombination technologies to allow a stable synthesis of organic compounds such as carboxylic acids, short-chain fatty acids and alcohol mainly from CO2 as a carbon source and with hydrogen as an "energy source" with high yield, both in anaerobic as well may also occur in aerobic processes.
Das technische Problem wird gelöst durch Bereitstellung einer trans- genen biologischen Zelle, besonders eines rekombinanten Mikroor- ganismus oder Teilen davon, welche eine Enzymausstattung enthält welche ausgewählt ist aus:The technical problem is solved by providing a transgenic biological cell, in particular a recombinant microorganism or parts thereof, which contains an enzyme equipment which is selected from:
(1 ) Hydrogenase-Aktivität, bevorzugt(1) Hydrogenase activity, preferred
(a) cytoplasmatische, bevorzugt NAD-reduzierende Hydrogenase-Aktivität, und/oder(a) cytoplasmic, preferably NAD-reducing hydrogenase activity, and / or
(b) membranständige Hydrogenase-Aktivität, bevorzugt zur Erzeugung eines Protonengradienten; und(b) membrane-bound hydrogenase activity, preferably for generating a proton gradient; and
(2) Enzymaktivität, geeignet zur Realisierung eines CO2- fixierenden Stoffwechselweges, ausgewählt aus:(2) Enzyme activity, suitable for realizing a CO2-fixing metabolic pathway, selected from:
Serin-Zyklus (SC), und zwar:Serine cycle (SC), namely:
Formyl-Tetrahydrofolat-Ligase-Aktivität; undFormyl-tetrahydrofolate ligase activity; and
reduktiver Tricarbonsäurezyklus (RTCC), und zwar:reductive tricarboxylic acid cycle (RTCC), namely:
Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität.Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
Zur Realisierung der Enzymausstattung enthält eine erfindungsgemäße Zelle, besonders im Genom, besonders in einer Expressionskassette und operativ verknüpft mit einem Promotor, mindestens ein Nucleinsäuremolekül mit einer Nucleotidsequenz, welche für mindestens eine der vorgenannten Enzymaktivitäten codiert.To realize the enzyme equipment, a cell according to the invention, especially in the genome, especially in an expression cassette and operably linked to a promoter, contains at least one nucleic acid molecule with a nucleotide sequence which codes for at least one of the aforementioned enzyme activities.
Gegenstand der Erfindung sind auch eine Expressionskassette und ein diese Expressionskassette enthaltender Vektor, welche geeignet sind, die Expression der erfindungsgemäß vorgesehenen Enzymaktivitäten in der Wirtszelle zu vermitteln. Bevorzugt enthält die Expressionskassette zur Transformation einer Wirtszelle:The invention also relates to an expression cassette and a vector containing this expression cassette, which are suitable for mediating the expression of the enzyme activities provided according to the invention in the host cell. Preferably, the expression cassette for transformation of a host cell contains:
(1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; und(1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Enzymaktivität ausgewählt aus: - Formyl-Tetrahydrofolat-Ligase-Aktivität;(2) at least one heterologous nucleic acid molecule encoding enzyme activity selected from: Formyl-tetrahydrofolate ligase activity;
- Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität.Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
Zur Herstellbarkeit des Endprodukts Lactat/Milchsäure sieht die Erfindung bevorzugt weiter vor, dass die Zelle, zusätzlich enthält:For the producibility of the end product lactate / lactic acid, the invention preferably further provides that the cell additionally contains:
(a) Lactat-Dehydrogenase,(a) lactate dehydrogenase,
(b) Lactat-Oxidoreduktase,(b) lactate oxidoreductase,
(c) mindestens einer Enzymaktivität des Methylglyoxal- wegs, und(c) at least one enzyme activity of the methylglyoxal path, and
(d) Aldehyd-Dehydrogenase(d) aldehyde dehydrogenase
In einer besonderen Variante der Erfindung kann zur Realisierung der CO2-Fixierung noch zusätzlich der Calvin-Benson-Bassham- Zyklus (CBB) realisiert sein, und zwar bevorzugt durch Expression mindestens einer heterologe Enzymaktivität, ausgewählt aus:In a particular variant of the invention, the Calvin-Benson-Bassham cycle (CBB) can additionally be realized for realizing the CO2 fixation, specifically by expressing at least one heterologous enzyme activity selected from:
Phosphoribulose-Kinase-Aktivität und Ribulosebisphosphat-Carboxylase-Aktivität,Phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity,
wobei bevorzugt zusätzlich die Enzymaktivität, ausgewählt aus:additionally preferably the enzyme activity selected from:
Fructose-Bisphosphatase-Aktivität und Fructosebisphosphat-Aldolase-AktivitätFructose bisphosphatase activity and fructose bisphosphate aldolase activity
in der Zelle nicht exprimiert oder gehemmt wird, Zur Realisierung dieser Variante der Erfindung wird bevorzugt eine transgene Zelle bereitgestellt, welche mindestens ein rekombinantes Nucleinsäuremolekül enthält mit einer Nucleotidsequenz, welche für mindestens eine Enzymaktivität codiert, welche ausgewählt ist aus:is not expressed or inhibited in the cell, To implement this variant of the invention, it is preferred to provide a transgenic cell containing at least one recombinant nucleic acid molecule having a nucleotide sequence encoding at least one enzyme activity selected from:
(1) Hydrogenase-Aktivität, bevorzugt cytoplasmatische und/oder membranständige Hydrogenase-Aktivität;(1) Hydrogenase activity, preferably cytoplasmic and / or membrane-bound hydrogenase activity;
(2) Enzymaktivität, geeignet zur Realisierung eines CO2- fixierenden Stoffwechselweges, ausgewählt aus:(2) Enzyme activity, suitable for realizing a CO2-fixing metabolic pathway, selected from:
Serin-Zyklus (SC) undSerine cycle (SC) and
- reduktiver Tricarbonsäurezyklus (RTCC);Reductive tricarboxylic acid cycle (RTCC);
und zusätzlich mindestens eine Enzymaktivität des CBB, ausgewählt aus:and additionally at least one enzyme activity of the CBB selected from:
Phosphoribulose-Kinase-Aktivität und Ribulosebisphosphat-Carboxylase-Aktivität;Phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity;
In dieser Variante enthält eine erfindungsgemäße Expressionskassette zur Transformation einer Wirtszelle zusätzlich: Phosphoribulo- se-Kinase-Aktivität und Ribulosebisphosphat-Carboxylase-Aktivität, wobei in der Expressionskassette, in einem die Expressionskassette enthaltenden Transformations-Vektor und in der zu transformieren- den Wirtszelle Nucleotidsequenzen, codierend für Enzymaktivität, ausgewählt aus Fructose-Bisphosphatase-Aktivität und Fructose- bisphosphat-Aldolase-Aktivität, nicht vorhanden oder nicht exprimiert oder diese Enzymaktivität gehemmt ist. In einer alternativen Variante der Erfindung ist in der erfindungsgemäßen Zelle zur Realisierung eines CO2-fixierenden Stoffwechselweges ausschließlich der Serin-Zyklus Serin-Zyklus (SC) und/oder der reduktive Tricarbonsäurezyklus (RTCC) realisiert; alternative CO2-fixierende Stoffwechselwege, und besonders der CBB sind unterdrückt oder gehemmt.In this variant, an expression cassette according to the invention for transforming a host cell additionally contains: phosphoribulosis kinase activity and ribulose bisphosphate carboxylase activity, nucleotide sequences being present in the expression cassette, in a transformation vector containing the expression cassette and in the host cell to be transformed coding for enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate aldolase activity, absent or absent or inhibiting this enzyme activity. In an alternative variant of the invention, only the serine cycle serine cycle (SC) and / or the reductive tricarboxylic acid cycle (RTCC) are realized in the cell according to the invention for realizing a CO2-fixing metabolic pathway; Alternative CO2 fixing pathways, and especially CBB, are suppressed or inhibited.
Gegenstand der Erfindung ist auch ein Verfahren zur biotechnologischen Herstellung von C2 - C6-Körpern als Produkt aus Kohlenstoffdioxid als Substrat und unter Assimilierung von Wasserstoff, Verwendung transgenen biologischen Zelle zur biotechnologischen Herstellung von Lactat oder Milchsäure sowie Alkohol aus Kohlenstoffdioxid.The invention also provides a process for the biotechnological production of C2-C6 bodies as product of carbon dioxide as substrate and assimilation of hydrogen, use of transgenic biological cell for the biotechnological production of lactate or lactic acid and alcohol from carbon dioxide.
Die Erfindung macht sich die überraschende Erkenntnis zunutze, dass in einer geeigneten transgenen beziehungsweise rekombinan- ten biologischen Zelle, besonders durch rekombinante Expression der vorgenannten Enzyme beziehungsweise Enzymaktivitäten die Fixierung von Kohlenstoffdioxid zur Herstellung von organischen Kohlenstoffverbindungen unter Verwendung eines Elektronendonors, vor allem Wasserstoff, mit hoher Ausbeute ermöglicht wird. Die Er- findung stellt somit Mittel zur Durchführung biotechnologischer Verfahren zu einer neuartigen hocheffektiven Umwandlung von insbesondere Wasserstoff und CO2 in einfach zu transportierende und nutzbare Treibstoffe, beispielsweise Alkohole, sowie chemische Grund- und Wertstoffe, beispielsweise Carbonsäuren und kurzketti- ge Fettsäuren dar. Die effektive Nutzung von CO2 als Kohlenstoffquelle ermöglicht so nicht nur die Realisierung CO2-neutraler Prozesse, sondern erlaubt durch die ubiquitäre Verfügbarkeit von CO2 auch eine unabhängige Standortwahl. Die vorliegende Erfindung macht sich vor allem die Verwendung von Wasserstoff als Elektro- nendonor als Energiequelle mikrobieller Prozesse zunutze. Der Wasserstoff steht dazu üblicherweise aus elektrochemischen Verfahren aus Wasser oder aus der Fermentation organischer Abfälle bereit.The invention makes use of the surprising finding that in a suitable transgenic or recombinant biological cell, in particular by recombinant expression of the abovementioned enzymes or enzyme activities, the fixation of carbon dioxide for the production of organic carbon compounds using an electron donor, especially hydrogen, with high Yield is made possible. The invention thus provides means for carrying out biotechnological processes for a novel, highly effective conversion of, in particular, hydrogen and CO.sub.2 into readily transportable and usable fuels, for example alcohols, as well as basic chemicals and valuable substances, for example carboxylic acids and short-chain fatty acids The use of CO2 as a carbon source thus not only enables the realization of CO2-neutral processes, but also allows an independent choice of location due to the ubiquitous availability of CO2. The present invention Above all, the use of hydrogen as an electron donor as an energy source for microbial processes is exploited. The hydrogen is usually prepared from electrochemical processes from water or from the fermentation of organic waste.
Es versteht sich, dass die Zelle mindestens eine der vorstehend definierten Enzymaktivitäten exprimiert. Bevorzugt exprimiert die Zelle zwei, mehrere und besonders bevorzugt alle der vorstehend definierten Enzymaktivitäten. Dazu ist das mindestens eine Nucleinsäure- molekül operativ verbunden mit einem Expressionssystem, das heißt mit einem Promotor oder Promotor-System. Es versteht sich, dass bevorzugt ein konstitutiver Promotor einsetzt wird. Zur Herstellung der erfindungsgemäßen Zelle wird der Wildtyp bevorzugt mit einem geeigneten Expressionsvektor, der die Expressionskassette enthält, transformiert.It is understood that the cell expresses at least one of the enzyme activities defined above. Preferably, the cell expresses two, more, and most preferably all of the enzyme activities defined above. For this purpose, the at least one nucleic acid molecule is operatively linked to an expression system, that is to say with a promoter or promoter system. It is understood that preferably a constitutive promoter is used. To produce the cell according to the invention, the wild type is preferably transformed with a suitable expression vector containing the expression cassette.
Die Transformation der erfindungsgemäßen Zellen und der Einbau der isolierten Nucleinsäuremoleküle erfolgt in an sich bekannter Weise, bevorzugt durch entsprechend geeignete Expressionsvektoren beziehungsweise in Verbindung mit entsprechenden Expressi- onskassetten, bevorzugt inklusive entsprechender Expressionsvektoren. Alternativ erfolgt die de novo-Synthese von Nucleinsäuremole- külen mit den gewünschten Nucleotidsequenzen, gegebenenfalls der kompletten Expressionskassette in einem geeigneten Synthesesystem.The transformation of the cells according to the invention and the incorporation of the isolated nucleic acid molecules takes place in a manner known per se, preferably by suitably suitable expression vectors or in conjunction with appropriate expression cassettes, preferably including appropriate expression vectors. Alternatively, the de novo synthesis of nucleic acid molecules takes place with the desired nucleotide sequences, optionally the complete expression cassette in a suitable synthesis system.
Bevorzugte Ausführungen der Erfindung sind:Preferred embodiments of the invention are:
Transgene biologische Zelle, enthaltend, vorzugsweise im Genom und insbesondere funktionell verknüpft mit einem Promotor: (1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; undTransgenic biological cell containing, preferably in the genome and in particular functionally linked to a promoter: (1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Enzymaktivität ausgewählt aus:(2) at least one heterologous nucleic acid molecule encoding enzyme activity selected from:
- Formyl-Tetrahydrofolat-Ligase-Aktivität;Formyl-tetrahydrofolate ligase activity;
Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität.Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
Die Transgene biologische Zelle, enthält, vorzugsweise im Genom und insbesondere funktionell verknüpft mit einem Promotor, gegebenenfalls zusätzlich: Phosphoribulose-Kinase-Aktivität und Ribu- losebisphosphat-Carboxylase-Aktivität, wobei Enzymaktivität, ausgewählt aus Fructose-Bisphosphatase-Aktivität und Fructosebis- phosphat-Aldolase-Aktivität, nicht exprimiert oder gehemmt wird.The transgenic biological cell contains, preferably in the genome and in particular functionally linked to a promoter, optionally in addition: phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity, wherein enzyme activity selected from fructose bisphosphatase activity and fructose bisphosphate- Aldolase activity, not expressed or inhibited.
In einer ersten Variante der Erfindung, worin zusätzlich der Calvin- Benson-Bassham-Zyklus (CBB) realisiert ist, enthält diese Zelle vor allem zur Steigerung des Synthesepotentials, zusätzlich mindestens ein heterologes Nucleinsäuremolekül, codierend für Enzymaktivität, ausgewählt aus:In a first variant of the invention, in which the Calvin- Benson-Bassham cycle (CBB) is additionally realized, this cell contains, in particular for increasing the synthesis potential, additionally at least one heterologous nucleic acid molecule coding for enzyme activity, selected from:
Fructose-6-Phosphat-Aldolase-Aktivität,Fructose 6-phosphate aldolase activity,
Glycerol-3-Phosphat-Dehydrogenase-Aktivität, Glycerolkinase-Aktivität, Glycerol-Dehydrogenase-Aktivität und Transaldolase-Aktivität. In einer bevorzugten Ausführung ist in der Zelle, bevorzugt an Stelle des CBB, der Serin-Zyklus realisiert. Dazu enthält die Zelle:Glycerol-3-phosphate dehydrogenase activity, glycerol kinase activity, glycerol dehydrogenase activity and transaldolase activity. In a preferred embodiment, the serine cycle is realized in the cell, preferably in place of the CBB. The cell contains:
(1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; und(1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Formyl-Tetrahydrofolat-Ligase-Aktivität.(2) at least one heterologous nucleic acid molecule encoding formyl tetrahydrofolate ligase activity.
In einer bevorzugten Variante weist die Zelle weiter die Enzymaktivität eines Glycin-Cleavage-Systems auf. Dazu enthält diese Zelle zusätzlich bevorzugt mindestens ein heterologes oder homologes Nuc- leinsäuremolekül codierend für diese Enzymaktivität.In a preferred variant, the cell further has the enzyme activity of a glycine cleavage system. For this purpose, this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
In einer bevorzugten Variante dieser Ausführung, weist die Zelle die Enzymaktivität ausgewählt aus:In a preferred variant of this embodiment, the cell has the enzyme activity selected from:
Serin-Glyoxylat-Aminotransferase-Aktivität, Hydroxypyruvat-Reduktase-Aktivität, Malat-Thiokinase-Aktivität,Serine-glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, malate thiokinase activity,
Maloyl-CoA-Lyase-Aktivität und Isocitrat-Lyase-Aktivität, undMaloyl-CoA lyase activity and isocitrate lyase activity, and
bevorzugt alle diese Aktivitäten auf.prefers all these activities.
In einer bevorzugten Variante enthält diese Zelle zusätzlich Formiat- Dehydrogenase-Aktivität zur Realisierung unter Verwendung des Serin-Zyklus. Dazu enthält diese Zelle zusätzlich bevorzugt mindestens ein heterologes oder homologes Nucleinsäuremolekül codierend für diese Enzymaktivität. In einer weiteren bevorzugten alternativen Ausfühunrg ist, bevorzugt an Stelle des CBB, der reduktive Tricarbonsäure-Zyklus realisiert. Dazu enthält die Zelle:In a preferred variant, this cell additionally contains formate dehydrogenase activity for realization using the serine cycle. In addition, this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity. In a further preferred alternative embodiment, preferably in place of the CBB, the reductive tricarboxylic acid cycle is realized. The cell contains:
(1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; und(1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Citrat-Lyase-Aktivität, Oxoglutarat- Oxidoreduktase-Aktivität und Fumarat-Reduktase- Aktivität;(2) at least one heterologous nucleic acid molecule encoding citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity;
bevorzugt sind Nucleinsäuremoleküle, codierend für alle diese Aktivitäten vorhanden, wobei zumindest ein Nucleinsäuremolekül eine heterologe Enzymaktivität codiert.Preferably, nucleic acid molecules encoding all of these activities are present, wherein at least one nucleic acid molecule encodes a heterologous enzyme activity.
In bevorzugten Varianten dieser Ausführungen enthält die Zelle: mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase- Aktivität, ausgewählt aus:In preferred variants of these embodiments, the cell contains: at least one nucleic acid molecule encoding hydrogenase activity selected from:
(1 )(a) membranständiger Hydrogenase-Aktivität, bevorzugt aus dem Mikroorganismus E. coli, ausgewählt aus Hydroge- nase hvaABC und Hydrogenase hybOCAB; und(1) (a) membrane-bound hydrogenase activity, preferably from the microorganism E. coli, selected from hydrogenase hvaABC and hydrogenase hybOCAB; and
(1 )(b) cytoplasmatischer NAD-reduzierender Hydrogenase- Aktivität, bevorzugt aus dem Mikroorganismus Ralstonia eutropha mit zumindest den Strukturgenen hoxFUYH.(1) (b) cytoplasmic NAD-reducing hydrogenase activity, preferably from the microorganism Ralstonia eutropha with at least the structural genes hoxFUYH.
Die Erfindung sieht in allen Varianten grundsätzlich vor, dass nur diejenigen Gene heterolog rekombiniert sind, die solche Enzymaktivitäten eines erfindungsgemäß identifizierten Stoffwechselwegs codie- ren, die nicht zur Enzymausstattung des Wildtyps der rekombinierten Zelle gehören. Die homologe Expression ist bevorzugt zur homologen Überexpression modifiziert. Alternativ ist in einer erfindungsgemäßen Zelle mindestens eine der beteiligten Enzymaktivitäten so- wohl homolog als auch heterolog exprimiert.The invention basically envisages in all variants that only those genes are recombined heterologously which encode such enzyme activities of a metabolic pathway identified according to the invention. which do not belong to the enzyme equipment of the wild type of the recombined cell. The homologous expression is preferably modified for homologous overexpression. Alternatively, in a cell according to the invention, at least one of the enzyme activities involved is expressed both homologously and heterologously.
Bevorzugt ist eine Zelle, die eine Lactat-Dehydrogenase-Aktivität exprimiert beziehungsweise aufweist. Dazu enthält diese Zelle zusätzlich bevorzugt mindestens ein heterologes oder homologes Nuc- leinsäuremolekül codierend für diese Enzymaktivität.Preferred is a cell that expresses or has a lactate dehydrogenase activity. For this purpose, this cell additionally preferably contains at least one heterologous or homologous nucleic acid molecule coding for this enzyme activity.
Zur Herstellbarkeit des Endprodukts Lactat/Milchsäure sieht die Erfindung bevorzugt vor, dass in der erfindungsgemäßen Zelle die bei der CO2-Assimilation gebildeten Zwischenprodukte überwiegend oder ausschließlich über die Aktivität einer Lactat-Dehydrogenase zu Lactat umsetzbar sind. Vorzugsweise ist in der erfindungsgemäßen Zelle dazu zusätzlich die Expression mindestens eines Lactat- Transporters realisiert. Alternativ sieht die Erfindung vor, dass die bei der CO2-Assimilation gebildeten Zwischenprodukte in der erfindungsgemäßen Zelle überwiegend oder ausschließlich über die Aktivität einer Lactat-Oxidoreduktase zu Lactat umsetzbar sind. Alterna- tiv sieht die Erfindung vor, dass die bei der CO2-Assimilation gebildeten Zwischenprodukte in der erfindungsgemäßen Zelle überwiegend oder ausschließlich über den Methylglyoxalweg zu Lactat umsetzbar sind. Alternativ sieht die Erfindung vor, dass die bei der CO2- Assimilation gebildeten Zwischenprodukte in der erfindungsgemäßen Zelle überwiegend oder ausschließlich über die Aktivität einer Alde- hyd-Dehydrogenase zu Lactat umsetzbar sind. Für enantiomerenreines Lactat ist in der erfindungsgemäßen Zelle die Expression von Aktivitäten eines enantiomerenselektiven Enzyms realisiert. Die Aktivität gegebenenfalls vorhandener homologer Enzyme mit mangelnder oder umgekehrter Stereospezifität und ge- gebenenfalls vorhandener Lactat-Racemase-Aktivität ist in der erfindungsgemäßen Zelle bevozugt gehemmt oder bevorzugt durch DeIe- tion der beteiligten Gene ausgeschaltet.For the producibility of the end product lactate / lactic acid, the invention preferably provides that in the cell according to the invention the intermediates formed during the CO2 assimilation can be converted predominantly or exclusively via the activity of a lactate dehydrogenase to lactate. Preferably, the expression of at least one lactate transporter is additionally realized in the cell according to the invention. Alternatively, the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of a lactate oxidoreductase to lactate. Alternatively, the invention provides that the intermediates formed in the course of CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the methylglyoxal route to lactate. Alternatively, the invention provides that the intermediates formed in the CO2 assimilation in the cell according to the invention can be converted predominantly or exclusively via the activity of an aldehyde dehydrogenase to lactate. For enantiomerically pure lactate, the expression of activities of an enantiomer-selective enzyme is realized in the cell according to the invention. The activity of optionally present homologous enzymes with lacking or reverse stereospecificity and, if present, lactate racemase activity is preferably inhibited in the cell according to the invention or preferably eliminated by de-ling of the genes involved.
Die Erfindung umfasst bevorzugt solche transgenen oder rekombi- nanten Zellen, die aus Bakterien, Cyanobakterien, Pilzen und Hefen ausgewählt oder abgeleitet sind. Bevorzugt ist die Zelle ausgewählt aus Bakterien der Gattungen Escherichia, Corynebacterium, Ralsto- nia, Clostridium, Pseudomonas, Bacillus, Lactobacillus und Lacto- coccus. Besonders bevorzugt ist die Zelle ausgewählt aus Bakterien der Spezies Escherichia coli, Corynebacterium glutamicum, Ralsto- nia eutropha, Pseudomonas putida, Lactobacillus plantarum und Clostridium acetobutylicum.The invention preferably includes those transgenic or recombinant cells selected or derived from bacteria, cyanobacteria, fungi and yeasts. The cell is preferably selected from bacteria of the genera Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Lactobacillus and Lactococcus. The cell is particularly preferably selected from bacteria of the species Escherichia coli, Corynebacterium glutamicum, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum and Clostridium acetobutylicum.
Die Erfindung umfasst auch solche biologischen Zellen, die ein synthetischer Mikroorganismus sind oder davon abgeleitet sind. Unter einer Zelle werden vorliegend auch Membranvesikel, Membrankom- partimente sowie Teile und Fragmente davon verstanden. Erfindungsgemäß bevorzugt befinden sich die Enzymaktivitäten im „Inneren" beziehungsweise Cytosol oder Cytoplasma der Zelle. Alternativ oder zusätzlich liegt mindestens eine der Enzymaktivitäten an einer Membran, besonders an einer Zellmembran assoziiert vor. Die Erfin- düng umfasst demgemäß auch oder Teile oder Fragmente eines erfindungsgemäßen transgenen Mikroorganismus, welche bevorzugt und in an sich bekannter Weise und unter Erhalt der Enzymaktivität an Trägerstrukturen assoziiert oder gebunden sind. Die Erfindung umfasst als „Zelle" auch einen Biokatalysator, worin die Enzymaktivitäten durch isolierte oder synthetisierte Enzymproteine mit den vorliegend angegebenen Aminosäuresequenzen realisiert sind. Dies geschieht vorzugsweise unter Umgehung zellulärer Ex- pressions- und Translationssysteme durch Inkorporation der Enzymproteine in die Zelle oder den Biokatalysator.The invention also includes those biological cells which are or derived from a synthetic microorganism. In the present case, a cell is also understood to mean membrane vesicles, membrane particles and parts and fragments thereof. According to the invention, the enzyme activities are preferably located in the "inner" or cytosol or cytoplasm of the cell.Alternatively or additionally, at least one of the enzyme activities is associated with a membrane, especially on a cell membrane.The invention accordingly also comprises or parts or fragments of a transgenic invention Microorganism, which are preferred and in a conventional manner and while maintaining the enzyme activity associated or bound to carrier structures. The invention also includes as a "cell" a biocatalyst in which the enzyme activities are realized by isolated or synthesized enzyme proteins having the amino acid sequences given herein, preferably by bypassing cellular expression and translation systems by incorporation of the enzyme proteins into the cell or biocatalyst.
Rekombinante Zellen, besonders rekombinante Mikroorganismen, welche die erfindungsgemäßen Gene enthalten und exprimieren, werden in an sich bekannter Weise durch übliche Rekombinati- onstechnologien hergestellt. Es versteht sich, dass die für die Rekombination ausgewählten Zellen oder Mikroorganismen gegenüber den gewünschten Stoffwechselendprodukten ausreichende Resistenz aufweisen. Bevorzugt sind Mikroorganismen, für die in der biotechnologischen Produktion standardisierte Kultivierungsbedingun- gen etabliert sind, sodass sich eine aufwändige Anpassung der etablierten biotechnologischen Prozessführung minimieren oder weitgehend vermeiden lässt.Recombinant cells, particularly recombinant microorganisms, which contain and express the genes according to the invention are produced in a manner known per se by customary recombination technologies. It is understood that the cells or microorganisms selected for recombination have sufficient resistance to the desired metabolic end products. Preference is given to microorganisms for which standardized cultivation conditions have been established in biotechnological production, so that elaborate adaptation of established biotechnological process management can be minimized or largely avoided.
Vom Umfang der Erfindung sind auch solche Zellen beziehungsweise Mikroorganismen und Zelllinien erfasst, die bereits in der nicht rekombinierten/transgenen Form zumindest eines oder mehrere der Gene beinhalten und exprimieren, die für eine der erfindungsgemäß vorgesehenen Enzymaktivitäten codieren. Es versteht sich, dass je nach Aktivität des vorhanden homologen Gens gegebenenfalls keine entsprechende Rekombination mit den analogen heterologen Genen erfolgen braucht. Gegebenenfalls werden an sich bekannte Maßnahmen zur Sicherstellung der homologen Expression beziehungsweise Überexpression des homologen Gens getroffen, um den erfindungsgemäßen Stoffwechsel weg vollständig zu realisieren. Je nach Enzymausstattung des Ausgangsorganismus kann es zusätzlich erforderlich sein, eventuell vorhandene konkurrierende Stoffwechselwege auszuschalten beziehungsweise zu unterdrücken. Dies kann durch an sich bekannte Maßnahmen erfolgen. Bevorzugte Ausgestaltungen solcher „enzymhemmender" Maßnahmen sind nachstehend beispielhaft für Mikroorganismen der Gattungen Escherichia und Ralstonia dargestellt. Zellen beziehungsweise Mikroorganismen, die entsprechende Deletionen zur Unterdrückung von konkurrierenden Stoffwechselwegen aufweisen, sind ebenfalls von der Erfindung umfasst.The scope of the invention also covers those cells or microorganisms and cell lines which, even in the non-recombined / transgenic form, contain and express at least one or more of the genes coding for one of the enzyme activities provided according to the invention. It is understood that, depending on the activity of the homologous gene present, it may not be necessary to recombine with the analogous heterologous genes. Optionally, measures known per se for ensuring the homologous expression or overexpression of the homologous gene are made in order to completely eliminate the metabolic pathway according to the invention. Depending on the enzyme equipment of the starting organism, it may additionally be necessary to eliminate or suppress any existing competing metabolic pathways. This can be done by measures known per se. Preferred embodiments of such "enzyme-inhibiting" measures are shown below by way of example for microorganisms of the genera Escherichia and Ralstonia.Cells or microorganisms which have corresponding deletions for suppressing competing metabolic pathways are likewise encompassed by the invention.
Eine bevorzugte Ausführung der Erfindung ist eine transgene beziehungsweise rekombinante E. coli Zelle, beispielsweise abgeleitet aus den Zelllinien K12. Eine alternative bevorzugte Ausführung der Erfindung ist eine transgene beziehungsweise rekombinante Ralstonia eutropha Zelle.A preferred embodiment of the invention is a transgenic or recombinant E. coli cell, for example derived from the cell lines K12. An alternative preferred embodiment of the invention is a transgenic or recombinant Ralstonia eutropha cell.
Neben den nachstehend bevorzugt genannten Nucleotidsequenzen und zugehörigen Aminosäuresequenzen können vergleichbare Gene aus dem Genom von weiteren Mikroorganismen in an sich bekannter Weise gefunden werden. Es wird auf die gängigen und an sich be- kannten Tools und Datenbanken der Bioinformatik verwiesen. Ist ein entsprechender Mikroorganismus, welcher die gewünschte Nucleo- tidsequenz in seinem Genom trägt, identifiziert, so können die entsprechenden Nucleinsäuremoleküle (DNA) mit den üblichen Verfahren daraus isoliert und amplifiziert werden.In addition to the preferred nucleotide sequences and corresponding amino acid sequences mentioned below, comparable genes from the genome of other microorganisms can be found in a manner known per se. Reference is made to the commonly used and well-known bioinformatics tools and databases. If a corresponding microorganism carrying the desired nucleotide sequence in its genome is identified, then the corresponding nucleic acid molecules (DNA) can be isolated therefrom and amplified by conventional methods.
Detaillierte Beschreibung der ErfindungDetailed description of the invention
Die Erfindung wird durch die Figuren 1 bis 16 näher erläutert. Fig. 1 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um die Reaktionen des Calvin-Benson-Bassham-Zyklus, beteiligt sind.The invention is explained in more detail by the figures 1 to 16. Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
Fig. 2 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um Teile des Calvin-Benson-Bassham-Zyklus, beteiligt sind.Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle.
Fig. 3 und 4 zeigen die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase, beteiligt sind.Figures 3 and 4 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase.
Fig. 5 und 6 zeigen die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase und die Schlüsselenzyme des Serin-Zyklus beteiligt sind.Figures 5 and 6 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by a formyl tetrahydrofolate ligase and the key enzymes of the serine cycle.
Fig. 7 und 8 zeigen die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um die Schlüsselenzyme des reduktiven Tricarbonsäure- zyklus (Citrat-Lyase, Oxoglutarat-Oxidoreduktase und Fumarat- Reduktase), beteiligt sind.FIGS. 7 and 8 show the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli, recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase).
Fig. 9 zeigt eine Flussverteilung, die das Synthesepotential für Lactat aus CO2 und Wasserstoff mit den in Fig. 1 dargestellten Reaktionsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Auf- nahmefluss an Wasserstoff (100%).FIG. 9 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 1. All rivers are hydrogen (100%) based on the intake flow.
Fig. 10 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 2 dargestellten Reakti- onsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 10 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
Fig. 11 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 3 dargestellten Reakti- onsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 11 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 3. All rivers are based on the uptake of hydrogen (100%).
Fig. 12 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 4 dargestellten Reaktionsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 12 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 4. All rivers are based on the uptake of hydrogen (100%).
Fig. 13 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 5 dargestellten Reaktionsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 13 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 5. All rivers are based on the uptake of hydrogen (100%).
Fig. 14 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 6 dargestellten Reaktionsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 14 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 6. All rivers are based on the uptake of hydrogen (100%).
Fig. 15 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 7 dargestellten Reaktionsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 15 shows a flux distribution which optimally utilizes the synthesis potential for lactate from CO 2 and hydrogen with the reaction steps illustrated in FIG. 7. All rivers are based on the uptake of hydrogen (100%).
Fig. 16 zeigt eine Flussverteilung, die das Synthesepotential für Lac- tat aus CO2 und Wasserstoff mit den in Fig. 8 dargestellten Reakti- onsschritten optimal ausnutzt. Alle Flüsse sind bezogen auf den Aufnahmefluss an Wasserstoff (100%).FIG. 16 shows a flux distribution which shows the synthesis potential for lactate from CO 2 and hydrogen with the reactivity shown in FIG. 8. optimally exploited. All rivers are based on the uptake of hydrogen (100%).
(1) Hydrogenase-Aktivität(1) Hydrogenase activity
Unter einer Hydrogenase-Aktivität, wird vorliegend die Fähigkeit zur Assimilation von elementarem Wasserstoff unter Bildung von Reduktionsäquivalenten verstanden.Hydrogenase activity is understood herein to mean the ability to assimilate elemental hydrogen to form reduction equivalents.
Die Erfindung sieht also bevorzugt vor, dass die Wirtszelle mit mindestens einem heterologen Hydrogenase-Operon ausgestattet wird. In einer weiteren Variante wird zusätzlich mindestens eine im WiId- typ der Zelle vorhandene homologe Hydrogenase-Aktivität expri- miert.The invention thus preferably provides that the host cell is equipped with at least one heterologous hydrogenase operon. In a further variant, at least one homologous hydrogenase activity existing in the WiId type of the cell is additionally expressed.
Zur Realisierung der Hydrogenase-Aktivität ist bevorzugt die Einbringung des pHG1 -Megaplasmids aus Ralstonia eutropha vorgesehen. Die Nucleotidsequenz des pHG1-Megaplasmids ist in Schwartz et al. 2003 (Schwartz E., Henne A., Cramm R, Eitinger T., Friedrich B., Gottschalk G. (2003). Complete Nucleotide Sequence of PHG1 : A Ralstonia eutropha H16 Megaplasmid Encoding Key Enzymes of H2- based Lithoautotrophy and Anaerobiosis. J Mol Biol 332, 369-383.) und unter Genbank Accession No. AY305378 publiziert; der Inhalt dieser Publikationen wird vollständig in den Offenbarungsgehalt der vorliegenden Beschreibung einbezogen. Das Megaplasmid ist 452 kbp groß und trägt 429 potentielle Gene. Darunter auch mindestens 41 Gene für Hydrogenase-Aktivität. In einer bevorzugten Variante ist deshalb vorgesehen, dass in die erfindungsgemäße Zelle ein ver- kürztes beziehungsweise modifiziertes Megaplasmid in die Zelle eingebracht ist, welches zumindest ein Hydrogenase-Operon des Me- gaplasids enthält. Die Erfindung sieht dazu bevorzugt die rekombinante Expression der cytoplasmatischen NAD-reduzierenden Hydrogenase-Aktivität aus Ralstonia eutropha vor. Dazu wird in der erfindungsgemäßen Zelle bevorzugt die Expression der Strukturgene hoxFUYH, vorzugsweise zusammen mit der an der Reifung des Enzyms beteiligten Gene, hypC1 , hypD1 , hypE1 und hypABF realisiert. Im Zusammenhang mit der Umsetzung mit dem Transkriptionssystem von Ralstonia ist bevorzugt die Expression des H2-Sensorsystems hoxA, hoxBC, hoxJ vorgesehen.For the realization of the hydrogenase activity, the introduction of the pHG1 megaplasmid from Ralstonia eutropha is preferably provided. The nucleotide sequence of the pHG1 megaplasmid is described in Schwartz et al. 2003 (Schwartz E., Hen A., Cramm R, Eitinger T., Friedrich B., Gottschalk G. (2003).) Complete Nucleotide Sequence of PHG1: A Ralstonia Eutropha H16 Megaplasmid Encoding Key Enzymes of H2-based Lithoautotrophy and Anaerobiosis. J Mol Biol 332, 369-383.) And under Genbank Accession no. AY305378 published; the content of these publications is fully incorporated in the disclosure of the present specification. The megaplasmid is 452 kbp in size and carries 429 potential genes. Including at least 41 genes for hydrogenase activity. In a preferred variant, it is therefore provided that a shortened or modified megaplasmide is incorporated into the cell according to the invention, which contains at least one hydrogenase operon of the gapase. The invention preferably provides for the recombinant expression of the cytoplasmic NAD-reducing hydrogenase activity from Ralstonia eutropha. For this purpose, the expression of the structural genes hoxFUYH, preferably together with the genes involved in the maturation of the enzyme, hypC1, hypD1, hypE1 and hypABF is preferably realized in the cell according to the invention. In connection with the reaction with the transcription system of Ralstonia, the expression of the H2 sensor system hoxA, hoxBC, hoxJ is preferred.
Bevorzugt stammt die heterologe Hydrogenase-Aktivität aus dem Mikroorganismus Ralstonia eutropha, besonders das Enzym NAD- Hydrogenase (EC 1.12.7.2), oder ist daraus abgeleitet. Die Erfindung macht sich dabei die Erkenntnis zunutze, dass vor allem die rekombinante Hydrogenase aus Ralstonia eutropha, vorteilhafterweise ver- gleichsweise aerotolerant ist und bereits selbst den universell verwendbaren Elektronenakzeptor NAD nutzt.The heterologous hydrogenase activity preferably originates from the microorganism Ralstonia eutropha, in particular the enzyme NAD hydrogenase (EC 1.12.7.2), or is derived therefrom. The invention makes use of the finding that, above all, the recombinant hydrogenase from Ralstonia eutropha, advantageously comparatively aerotolerant and already uses even the universally usable electron acceptor NAD.
Nach dem bisherigen Stand der Erkenntnis ist die NAD-Hydrogenase (EC 1.12.7.2) aus Ralstonia eutropha ein Heterotetramer aus 4 Untereinheiten. Bevorzugte, diese Untereinheiten codierenden Nucleo- tidsequenzen sind SEQ ID NO: 1 für Untereinheit hoxF, SEQ ID NO: 3 für Untereinheit hoxU, SEQ ID NO: 5 für Untereinheit hoxY und SEQ ID NO: 7 für Untereinheit hoxH. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 2, 4, 6, und 8 auf.According to the current state of knowledge, the NAD hydrogenase (EC 1.12.7.2) from Ralstonia eutropha is a heterotetramer of 4 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 1 for hoxF subunit, SEQ ID NO: 3 for hoxU subunit, SEQ ID NO: 5 for hoxY subunit, and SEQ ID NO: 7 for hoxH subunit. The enzyme protein thus preferably has the amino acid sequences SEQ ID NO: 2, 4, 6, and 8.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei, mindestens drei oder bevorzugt alle heterologe Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus: a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 1 , 3, 5, 7 enthalten oder daraus bestehen;The invention thus preferably relates to a cell which contains at least one, preferably at least two, at least three or preferably all heterologous nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1, 3, 5, 7;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 2, 4, 6, 8 enthalten oder dar- aus bestehen, codieren; undb) nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 2, 4, 6, 8; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und be- vorzugt eine Hydrogenase-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a hydrogenase activity.
Die Erfindung sieht zur Expression der Hydrogenase-Aktivität bevorzugt weiter vor: die Expression mindestens eines an der Reifung der Hydrogenase-Aktivität beteiligten Proteins. Bevorzugt sind die Strukturgene ausgewählt aus: codierenden Nucleotidsequenzen SEQ ID NO: 9 für hypC1. SEQ ID NO: 11 für hvpD1 und SEQ ID NO: 13 für hvpEL Für die Proteine hypA, hypB und hvpF sind bevorzugt die codierenden Nucleotidsequenzen SEQ ID NO: 15 für hypA1 , SEQ ID NO: 17 für hvpB1 und SEQ ID NO: 19 für hypF1 vorgesehen. Alternativ sind SEQ ID NO: 21 für hypA2, SEQ ID NO: 23 für hypB2 und SEQ ID NO: 25 für hypF2. Die für die Reifung relevanten Proteine weisen also bevorzugt die entsprechenden Aminosäuresequenzen SEQ ID NO: 10, 12, 14, sowie 16, 18, 20 und/oder 22, 24, 26 auf.The invention preferably further provides for the expression of the hydrogenase activity: the expression of at least one protein involved in the maturation of the hydrogenase activity. Preferably, the structural genes are selected from: nucleotide sequences encoding SEQ ID NO: 9 for hypC1. SEQ ID NO: 11 for hvpD1 and SEQ ID NO: 13 for hvpEL For the proteins hypA, hypB and hvpF, preferably the coding nucleotide sequences SEQ ID NO: 15 for hypA1, SEQ ID NO: 17 for hvpB1 and SEQ ID NO: 19 for hypF1 provided. Alternatively, SEQ ID NO: 21 for hypA2, SEQ ID NO: 23 for hypB2, and SEQ ID NO: 25 for hypF2. The proteins relevant for the maturation thus preferably have the corresponding amino acid sequences SEQ ID NO: 10, 12, 14, as well as 16, 18, 20 and / or 22, 24, 26.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche außerdem mindestens ein, bevorzugt mindestens zwei, drei, vier, fünf, sechs, sieben, acht oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus: a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 enthalten oder daraus bestehen;The invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, three, four, five, six, seven, eight or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 9, 11, 13, 15, 17, 19, 21, 23, 25;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24,b) nucleic acid molecules which are suitable for amino acid molecules which have the sequences SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24,
26 enthalten oder daraus bestehen, codieren; und26 contain or consist of encode; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt ein Reifungsprotein für Hydrogenase-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably encode a maturation protein for hydrogenase activity.
Die Erfindung sieht im Zusammenhang mit der Expression der Hydrogenase-Aktivität bevorzugt weiter vor: die Expression mindes- tens eines der am H2-Sensor-System beteiligten Proteine. Bevorzugt ist das H2-Sensor-Protein ausgewählt aus: codierenden Nucleotid- sequenzen SEQ ID NO: 27 für hoxA, SEQ ID NO: 29 für Untereinheit hoxB und SEQ ID NO: 31 für Untereinheit hoxC von hoxBC sowie SEQ ID NO: 33 für hoxJ. Die H2-Sensor-Proteine weisen also bevor- zugt die entsprechenden Aminosäuresequenzen SEQ ID NO: 28, 30, 32 und 34 auf. Das H2-Sensorsystem (hoxA, hoxBC, hoxJ) wird bevorzugt nur dann realisiert, wenn ein Ralstonia-eigenes Transkriptionssystem verwendet wird.The invention preferably further provides in connection with the expression of the hydrogenase activity: the expression of at least one of the proteins involved in the H2 sensor system. Preferably, the H2 sensor protein is selected from: coding nucleotide sequences SEQ ID NO: 27 for hoxA, SEQ ID NO: 29 for hoxB subunit and SEQ ID NO: 31 for hoxC subunit of hoxBC and SEQ ID NO: 33 for hoxJ , The H2 sensor proteins therefore preferably have the corresponding amino acid sequences SEQ ID NO: 28, 30, 32 and 34. The H2 sensor system (hoxA, hoxBC, hoxJ) is preferably only realized if a Ralstonia own transcription system is used.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche außerdem mindestens ein, bevorzugt mindestens zwei, mindestens drei oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus: a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 27, 29, 31 , 33 enthalten oder daraus bestehen;The invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 27, 29, 31, 33;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 28, 30, 32, 34 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 28, 30, 32, 34; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und be- vorzugt ein H2-Sensor-Protein codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an H 2 sensor Encode protein.
Zur Realisierung der Hydrogenase-Aktivität sieht die Erfindung alternativ oder bevorzugt zusätzlich die Expression der Aktivität einer membranständigen Hydrogenase aus E. coli vor. Dies ist besonders das Enzym Hydrogenase hyaABC und das Enzym Hydrogenase hvbOCAB. In dieser Variante sieht die Erfindung vor, dass, alternativ oder bevorzugt zusätzlich, die Aktivität einer membranständigen Hydrogenase exprimiert wird. Bevorzugt stammt die Hydrogenase- Aktivität aus dem Mikroorganismus E. coli, besonders das Enzym Hydrogenase hyaABC oder Hydrogenase hvbOCAB, oder es ist da- von abgeleitet.To realize the hydrogenase activity, the invention provides alternatively or preferably additionally the expression of the activity of a membrane-bound hydrogenase from E. coli. This is especially the enzyme hydrogenase hyaABC and the enzyme hydrogenase hvbOCAB. In this variant, the invention provides that, alternatively or preferably in addition, the activity of a membrane-bound hydrogenase is expressed. Preferably, the hydrogenase activity is derived from the microorganism E. coli, especially the enzyme hydrogenase hyaABC or hydrogenase hvbOCAB, or it is derived therefrom.
In einer ersten Alternative dieser Variante ist die Expression der membranständigen Hydrogenase hvaABC realisiert. Nach dem bisherigen Stand der Erkenntnis ist die Hydrogenase hvaABC aus E. coli ein Heteromer aus 3 Untereinheiten. Diese Untereinheiten codie- renden Nucleotidsequenzen sind SEQ ID NO: 137 für Untereinheit hvaA, SEQ ID NO: 139 für Untereinheit hyaB und SEQ ID NO: 141 für Untereinheit hyaC. Das Enzymprotein Hydrogenase hvaABC weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 138, 140 und 142 auf.In a first alternative of this variant, the expression of the membrane-bound hydrogenase hvaABC is realized. According to the current state of knowledge, the hydrogenase hvaABC from E. coli is a heteromer of 3 subunits. These subunit-encoding nucleotide sequences are SEQ ID NO: 137 for hvaA subunit, SEQ ID NO: 139 for hyaB subunit, and SEQ ID NO: 141 for hyaC subunit. The enzyme protein hydrogenase hvaABC Thus, it preferably has the amino acid sequences SEQ ID NO: 138, 140 and 142.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche außerdem mindestens ein, bevorzugt mindestens zwei, oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 137, 139, 141 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 137, 139, 141;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 138, 140, 142 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 138, 140, 142; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Hydrogenase-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
Analog dazu in einer weiteren Alternative ist, bevorzugt zusätzlich, die Expression der membranständigen Hydrogenase hybOCAB realisiert. Nach dem bisherigen Stand der Erkenntnis ist die Hydrogena- se hybOCAB aus E. coli ein Heteromer aus 4 Untereinheiten. Diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 149 für Untereinheit hybO, SEQ ID NO: 155 für Untereinheit hybC, und SEQ ID NO: 151 für Untereinheit hybA und SEQ ID NO: 153 für Untereinheit hybB. Das Enzymprotein Hydrogenase hybOCAB weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 150, 156, 152 und 154 auf. Die Erfindung betrifft somit bevorzugt eine Zelle, welche außerdem mindestens ein, bevorzugt mindestens zwei, mindestens drei oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:Analogously, in another alternative, preferably additionally, the expression of the membrane-bound hydrogenase hybOCAB is realized. According to the current state of knowledge, the hydrogenase hybOCAB from E. coli is a heteromer of 4 subunits. Nucleotide sequences encoding these subunits are SEQ ID NO: 149 for subunit hybO, SEQ ID NO: 155 for subunit hybC, and SEQ ID NO: 151 for subunit hybA and SEQ ID NO: 153 for subunit hybB. The enzyme protein hydrogenase hybOCAB thus preferably has the amino acid sequences SEQ ID NO: 150, 156, 152 and 154. The invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO:a) nucleic acid molecules which have the sequences SEQ ID NO:
149, 151 , 153, 155 enthalten oder daraus bestehen;149, 151, 153, 155 contain or consist of;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 150, 152, 154, 156 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 150, 152, 154, 156; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Hydrogenase-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably encode a hydrogenase activity.
Im Zusammenhang mit der Verwendung der membranständigen Hydrogenasen aus E. coli ist auch die Expression der zugehörigen Chaperone hyaD, hyaE und hyaF beziehungsweise hybD, hybE, hybF und hybG zumindest teilweise realisiert. Die die Chaperone codierenden Nucleotidsequenzen sind SEQ ID NO: 143 für hyaD, SEQ ID NO: 145 für hyaE und SEQ ID NO: 147 für hyaF. Die Chaperone weisen also bevorzugt die Aminosäuresequenzen SEQ ID NO: 144, 146 und 148 auf. Die Chaperon-Gene hyaE und hyaF sind optional und brauchen nicht exprimiert zu werden.In connection with the use of membrane-bound hydrogenases from E. coli, the expression of the associated chaperones hyaD, hyaE and hyaF or hybD, hybE, hybF and hybG is also at least partially realized. The nucleotide sequences encoding the chaperones are SEQ ID NO: 143 for hyaD, SEQ ID NO: 145 for hyaE, and SEQ ID NO: 147 for hyaF. The chaperones thus preferably have the amino acid sequences SEQ ID NO: 144, 146 and 148. The chaperone genes hyaE and hyaF are optional and need not be expressed.
Die die Chaperone hybD, hybE. hybF und hybG codierenden Nucle- otidsequenzen sind SEQ ID NO: 157 für hybP, SEQ IP NO: 159 ITy1 b_E, SEQ IP NO: 161 für hybF und SEQ IP NO: 163 für hybG. Pie Chaperone weisen also bevorzugt die Aminosäuresequenzen SEQ ID NO: 158, 160, 162 und 164 auf.The chaperones hybD, hybE. HybF and hybG coding nucleotide sequences are SEQ ID NO: 157 for hybP, SEQ IP NO: 159 ITy 1 b_E, SEQ IP NO: 161 for hybF and SEQ IP NO: 163 for hybG. Pie Thus, chaperones preferably have the amino acid sequences SEQ ID NO: 158, 160, 162 and 164.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche außerdem mindestens ein, bevorzugt mindestens zwei, mindestens drei oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which additionally contains at least one, preferably at least two, at least three or preferably all nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 143, 145, 147, 157, 159, 161 , 163 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 143, 145, 147, 157, 159, 161, 163;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 144, 146, 148, 158, 160, 162, 164 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 144, 146, 148, 158, 160, 162, 164; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, be- vorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt Hydrogenase-Chaperone codieren.c) nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably code for hydrogenase chaperones.
(2a) Calvin-Benson-Bassham-Zyklus (CBB)(2a) Calvin-Benson-Bassham Cycle (CBB)
Unter einer Enzymaktivität eines am CBB beteiligten Enzyms wird vorliegend die Fähigkeit zur Umsetzung eines Zwischenprodukts des CBB, Calvin-Zyklus oder reduktivem Pentosephosphatweg, als Substrat in ein anderes Zwischenprodukt des Zyklus als Produkt der Enzymreaktion verstanden. Nach dem bisherigen Stand der Erkenntnis sind 11 Enzyme am CBB beteiligt. Allgemein ist der CBB in drei Ab- schnitte einteilbar: eigentliche Kohlenstoff-Fixierung (Carboxylie- rung), Reduktionsphase (pro drei Zyklen wird ein C3-Körper als Produkt abgegeben) und Regenerationsphase des Kohlenstoff- Akzeptors Ribulose-1 ,5-bisphosphat.By enzymatic activity of an enzyme involved in CBB herein is meant the ability to react an intermediate of the CBB, Calvin cycle or reductive pentose phosphate pathway as a substrate into another intermediate product of the cycle as a product of the enzyme reaction. According to the current state of knowledge, 11 enzymes are involved in CBB. In general, the CBB can be divided into three sections: actual carbon fixation (carboxylation) tion), reduction phase (a C3 body is released as product per three cycles) and regeneration phase of the carbon acceptor ribulose-1, 5-bisphosphate.
Erfindungsgemäß werden als Schlüsselenzymaktivitäten des CBB die Aktivitäten einer Phosphoribulose-Kinase (Phosphoribulokinase) und einer Ribulosebisphosphat-Carboxylase angesehen. Die Erfindung sieht zur Realisierung des CBB in der erfindungsgemäßen Zelle vor: die gegebenenfalls heterologe Expression von zumindest einer Phosphoribulose-Kinase-Aktivität und einer Ribulose- bisphosphat-Carboxylase-Aktivität.According to the invention, the activities of a phosphoribulose kinase (phosphoribulokinase) and a ribulose bisphosphate carboxylase are considered as key enzyme activities of CBB. The invention provides for the realization of the CBB in the cell according to the invention: the optionally heterologous expression of at least one phosphoribulose kinase activity and one ribulose bisphosphate carboxylase activity.
Fig. 1 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat in Escherichia coli, rekombinant erweitert um die Reaktionen des Calvin-Benson-Bassham-Zyklus, beteiligt sind.Figure 1 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate in Escherichia coli recombinantly extended by the reactions of the Calvin-Benson-Bassham cycle.
Im Zusammenhang mit der Realisierung der Phosphoribulose- Kinase-Aktivität und Ribulosebisphosphat-Carboxylase-Aktivität sieht die Erfindung vor allem vor, dass in der erfindungsgemäßen Zelle die Expression von Fructose-Bisphosphatase-Aktivität und/oder von Fructosebisphosphat-Aldolase-Aktivität durch Deletion der diese Ak- tivität codierenden Nucleotidsequenzen ausgeschaltet ist.In connection with the realization of the phosphoribulose kinase activity and ribulose bisphosphate carboxylase activity, the invention provides, above all, that in the cell according to the invention the expression of fructose bisphosphatase activity and / or of fructose bisphosphate aldolase activity by deletion of these Activity-encoding nucleotide sequences is switched off.
Die Erfindung sieht dazu vor allem vor, dass zur CO2-Assimilation überwiegend oder ausschließlich der Calvin-Benson-Bassham- Zyklus (CBB) realisiert wird, wobei in der erfindungsgemäßen Zelle mindestens eine am CBB beteiligte Enzymaktivitäten in ausreichen- dem Umfang exprimiert werden. Es ist bevorzugt vorgesehen, dass die Expression von Aktivitäten von Enzymen, ausgewählt aus: Phosphoribulose-Kinase-Aktivität (Phosphoribulokinase) und Ribulosebisphosphat-Carboxylase-AktivitätThe invention provides, above all, for predominantly or exclusively the Calvin-Benson-Bassham cycle (CBB) to be implemented for CO2 assimilation, wherein at least one enzyme activity involved in CBB is sufficiently expressed in the cell according to the invention. It is preferably provided that the expression of activities of enzymes selected from: Phosphoribulose kinase activity (phosphoribulokinase) and ribulose bisphosphate carboxylase activity
und/oder damit vergleichbare Aktivitäten realisiert ist.and / or comparable activities are realized.
Die Erfindung sieht dazu weiter vor, dass der Calvin-Benson- Bassham-Zyklus (CBB) unter Vermeidung, Hemmung beziehungsweise Deletion mindestens einer, bevorzugt beider, gegebenenfalls im Wildtyp der Zelle vorhandenen Enzymaktivität, ausgewählt aus:The invention further provides that the Calvin-Benson-Bassham cycle (CBB), while avoiding, inhibiting or deleting at least one, preferably both, enzyme activity optionally present in the wild type of the cell, is selected from:
Fructose-Bisphosphatase-Aktivität und Fructosebisphosphat-Aldolase-AktivitätFructose bisphosphatase activity and fructose bisphosphate aldolase activity
realisiert wird, falls solche Enzymaktivitäten im Wildtyp homolog exprimiert werden. Dies ist vor allem im Zusammenhang mit der Verwendung einer Wirtszelle aus Ralstonia besonders von Ralstonia eutropha beziehungsweise des Megaplasmids pHG 1 aus Ralstonia eutropha zur Transformation von Wirtszellen relevant.is realized, if such enzyme activities are expressed homologously in the wild type. This is particularly relevant in connection with the use of a host cell from Ralstonia especially of Ralstonia eutropha or the megaplasmid pHG 1 from Ralstonia eutropha for the transformation of host cells.
In einer Variante sieht die Erfindung vor, dass der CBB unter Hemmung beziehungsweise Deletion einer Fructose-Bisphosphatase- Aktivität realisiert ist. Dies geschieht erfindungsgemäß bevorzugt durch Hemmung der Expression und/oder Deletion mindestens einer diese Enzymaktivität codierenden Nucleotidsequenz beziehungswei- se mindestens eines der Gene. In Verbindung mit der Verwendung einer rekombinanten E. coli Zelle sieht die Erfindung dazu bevorzugt vor:In a variant, the invention provides that the CBB is realized by inhibiting or deleting a fructose-bisphosphatase activity. This is preferably done according to the invention by inhibiting the expression and / or deletion of at least one nucleotide sequence coding for this enzyme activity or at least one of the genes. In connection with the use of a recombinant E. coli cell, the invention preferably provides for this:
die Hemmung der Aktivität der Fructose-Bisphosphatase I fbp_ bevorzugt durch Deletion mindestens des Gens für fbj), dar- gestellt durch die SEQ ID NO: 77 oder codierend für die Ami- nosäuresequenz SEQ ID NO: 78, und alternativ oder bevorzugt zusätzlichthe inhibition of the activity of the fructose bisphosphatase I fbp_ preferably by deletion of at least the gene for fbj), represented by the SEQ ID NO: 77 or coding for the amide Nosäuresequenz SEQ ID NO: 78, and alternatively or preferably in addition
die Hemmung der Aktivität der Fructose-Bisphosphatase Il glpX bevorzugt durch Deletion mindestens des Gens für glpX, dargestellt durch die SEQ ID NO: 79 oder codierend für diethe inhibition of the activity of the fructose bisphosphatase II glpX preferably by deletion of at least the gene for glpX, represented by the SEQ ID NO: 79 or coding for the
Aminosäuresequenz SEQ ID NO: 80Amino acid sequence SEQ ID NO: 80
In einer, alternativen oder bevorzugt zusätzlichen, Variante sieht die Erfindung vor, dass der CBB unter Hemmung beziehungsweise Deletion einer Fructosebisphosphat-Aldolase-Aktivität realisiert ist. Dies geschieht erfindungsgemäß bevorzugt durch Hemmung der Expression und/oder Deletion mindestens einer diese Enzymaktivität codierenden Nucleotidsequenz beziehungsweise mindestens eines der Gene. In Verbindung mit der Verwendung einer rekombinanten E. coli Zelle sieht die Erfindung dazu bevorzugt alternativ oder bevor- zugt zusätzlich vor:In an alternative or preferred additional variant, the invention provides that the CBB is realized by inhibiting or deleting a fructose bisphosphate aldolase activity. This is done according to the invention preferably by inhibiting the expression and / or deletion of at least one nucleotide sequence encoding this enzyme activity or at least one of the genes. In connection with the use of a recombinant E. coli cell, the invention preferably additionally or alternatively additionally provides for this:
die Hemmung der Fructosebisphosphat-Aldolase Il-Aktivität fbaA durch Deletion mindestens des Gens für fbaA, dargestellt durch die SEQ ID NO: 81 oder codierend für die Aminosäuresequenz SEQ ID NO: 82, und alternativ oder bevorzugt zusätzlichthe inhibition of fructose bisphosphate aldolase II activity fbaA by deleting at least the gene for fbaA represented by SEQ ID NO: 81 or coding for the amino acid sequence SEQ ID NO: 82, and alternatively or preferably additionally
die Hemmung der Fructosebisphosphat-Aldolase I-Aktivität fbaB durch Deletion mindestens des Gens für fbaB, dargestellt durch die SEQ ID NO: 83 oder codierend für die Aminosäuresequenz SEQ ID NO: 84.the inhibition of fructose bisphosphate aldolase I activity fbaB by deletion of at least the gene for fbaB represented by SEQ ID NO: 83 or coding for the amino acid sequence SEQ ID NO: 84.
Bevorzugt stammt die Phosphoribulose-Kinase-Aktivität aus dem Mikroorganismus Ralstonia eutropha, besonders das Enzym Phosphoribulose-Kinase cbbPp, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 35. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 36 auf.Preferably, the phosphoribulose kinase activity is from the microorganism Ralstonia eutropha, especially the enzyme Phosphoribulose kinase cbbPp, or is derived from it. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 35. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 36.
Bevorzugt stammt die Ribulosebisphosphat-Carboxylase-Aktivität aus dem Mikroorganismus Ralstonia eutropha, besonders das Enzym Ribulosebisphosphat-Carboxylase cbbSp/cbbLp, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist die Ribulo- sebisphosphat-Carboxylase aus 2 Untereinheiten aufgebaut. Bevor- zugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 37 für Untereinheit cbbLp und SEQ ID NO: 39 für Untereinheit cbbSp. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 40 und 42 auf.Preferably, the ribulose bisphosphate carboxylase activity is derived from the microorganism Ralstonia eutropha, especially the enzyme ribulose bisphosphate carboxylase cbbSp / cbbLp, or is derived therefrom. According to the current state of knowledge, the ribulose bisphosphate carboxylase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 37 for subunit cbbLp and SEQ ID NO: 39 for subunit cbbSp. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 40 and 42.
In einer Variante sieht die Erfindung bevorzugt vor, dass zur Reali- sierung des CBB in der erfindungsgemäßen Zelle in der Zelle das pHG1-Megaplasmid aus Ralstonia eutropha vorliegt und dort, neben des Hydrogenase-Operons besonders die Phosphoribulose-Kinase- Operon und/oder die Ribulosebisphosphat-Carboxylase-Operon exprimiert werden. Dabei ist gemäß der Erfindung vorgesehen, ein verkürztes oder modifiziertes pHG1-Megaplasmid einzusetzen, worin die Operons, codierend für Fructose-Bisphosphatase-Aktivität und Fructosebisphosphat-Aldolase-Aktivität, deletiert oder unterdrückt sind.In a variant, the invention preferably provides for the realization of the CBB in the cell according to the invention in the cell, the pHG1 megaplasmid from Ralstonia eutropha is present and there, in addition to the hydrogenase operon especially the phosphoribulose kinase operon and / or the Ribulose bisphosphate carboxylase operon are expressed. It is provided according to the invention to use a truncated or modified pHG1 megaplasmid, wherein the operons coding for fructose bisphosphatase activity and fructose bisphosphate aldolase activity are deleted or suppressed.
Die Erfindung betrifft bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei oder bevorzugt alle Nucleinsäuremolekü- Ie enthält, welche ausgewählt sind aus der Gruppe bestehend aus: a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 35, 37, 39 enthalten oder daraus bestehen;The invention preferably relates to a cell which contains at least one, preferably at least two or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 35, 37, 39;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 36, 38, 40 enthalten oder dar- aus bestehen, codieren; undb) nucleic acid molecules coding for amino acid molecules which contain or consist of the sequences SEQ ID NO: 36, 38, 40; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und be- vorzugt eine Phosphoribulose-Kinase-Aktivität und/oder eine Ribulosebisphosphat-Carboxylase-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a phosphoribulose kinase activity and / or encode a ribulose bisphosphate carboxylase activity.
In diesen Varianten ist bevorzugt vorgesehen, dass die Expression einer Fructose-6-Phosphat-Aldolase-Aktivität realisiert ist. Bevorzugt stammt die Fructose-6-Phosphat-Aldolase-Aktivität aus dem Mikro- Organismus E. coli, besonders ist es das Enzym Fructose-6- Phosphat-Aldolase fsaA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 85. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 86 auf.In these variants, it is preferably provided that the expression of a fructose-6-phosphate aldolase activity is realized. Preferably, the fructose-6-phosphate aldolase activity is derived from the microorganism E. coli, especially the enzyme fructose-6-phosphate aldolase fsaA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 85. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 86.
Alternativ bevorzugt ist das Enzym Fructose-6-Phosphat-Aldolase fsaB, oder es ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 87. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 88 auf.Alternatively, preferably, the enzyme is fructose 6-phosphate aldolase fsaB, or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 87. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 88.
In diesen Varianten ist bevorzugt weiter vorgesehen, dass die Ex- pression einer Glycerol-3-Phosphat-Dehydrogenase-Aktivität realisiert ist. Bevorzugt stammt die Glycerol-3-Phosphat-Dehydrogenase- Aktivität aus dem Mikroorganismus E. coli, besonders ist das Enzym Glycerol-3-Phosphat-Dehydrogenase gpsA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 89. Das Enzymprotein weist also bevorzugt die Amino- säuresequenz SEQ ID NO: 90 auf.In these variants, it is preferably further provided that the expression of a glycerol-3-phosphate dehydrogenase activity is realized. Preferably, the glycerol-3-phosphate dehydrogenase Activity from the microorganism E. coli, especially the enzyme glycerol-3-phosphate dehydrogenase gpsA, or is derived from it. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 89. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 90.
Alternativ bevorzugt ist das Enzym Glycerol-3-Phosphat- Dehydrogenase qlpABC, oder es ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist diese Glycerol-3-Phosphat- Dehydrogenase aus 3 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 91 für Untereinheit qlpA, SEQ ID NO: 93 für Untereinheit qlpB und SEQ ID NO: 95 für Untereinheit qlpC. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 92, 94 und 96 auf.Alternatively, the enzyme glycerol-3-phosphate dehydrogenase qlpABC is preferred, or it is derived therefrom. According to the current state of knowledge, this glycerol-3-phosphate dehydrogenase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 91 for subunit qlpA, SEQ ID NO: 93 for subunit qlpB and SEQ ID NO: 95 for subunit qlpC. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 92, 94 and 96.
Alternativ bevorzugt ist das Enzym Glycerol-3-Phosphat- Dehydrogenase qlpD, oder es ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 97. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 98 auf.Alternatively, the enzyme glycerol-3-phosphate dehydrogenase qlpD is preferred, or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 97. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 98.
In diesen Varianten ist bevorzugt weiter vorgesehen, dass die Ex- pression einer Glycerol-Kinase-Aktivität realisiert ist. Bevorzugt stammt die Glycerol-Kinase-Aktivität aus dem Mikroorganismus E. coli, besonders ist das Enzym Glycerol-Kinase glpK, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 99. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 100 auf.In these variants, it is further preferred that the expression of a glycerol kinase activity is realized. Preferably, the glycerol kinase activity is derived from the microorganism E. coli, especially the enzyme glycerol kinase glpK, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 99. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 100.
In diesen Varianten ist bevorzugt weiter vorgesehen, dass die Expression einer Glycerol-Dehydrogenase-Aktivität realisiert ist. Bevor- zugt stammt die Glycerol-Dehydrogenase-Aktivität aus dem Mikroorganismus E. coli, besonders ist das Enzym Glycerol-Dehydrogenase gldA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 101. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 102 auf.In these variants, it is further preferred that the expression of a glycerol dehydrogenase activity is realized. Before- zugt the glycerol dehydrogenase activity comes from the microorganism E. coli, especially the enzyme glycerol dehydrogenase gldA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 101. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 102.
In diesen Varianten ist schließlich bevorzugt weiter vorgesehen, dass die Expression einer Transaldolase-Aktivität realisiert ist. Bevorzugt stammt die Transaldolase-Aktivität aus dem Mikroorganismus E. coli, besonders ist das Enzym die Transaldolase talA, oder ist daraus ab- geleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 103. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 104 auf.Finally, in these variants, it is further preferred that the expression of a transaldolase activity is realized. The transaldolase activity preferably originates from the microorganism E. coli, in particular the enzyme is the transaldolase talA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 103. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 104.
Alternativ bevorzugt ist das Enzym die Transaldolase talB, oder es ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 105. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 106 auf.Alternatively, preferably, the enzyme is the transaldolase talB, or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 105. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 106.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei, drei, vier, fünf, sechs, sieben, acht, neun, zehn oder bevorzugt alle Nucleinsäuremoleküle enthält, wel- che ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 include or consist of encode; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, be- vorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Enzymaktivität, ausgewählt aus:c) nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an enzyme activity selected from:
Fructose-6-Phosphat-Aldolase-Aktivität, Glycerol-3-Phosphat-Dehydrogenase-Aktivität, Glycerolkinase-Aktivität,Fructose-6-phosphate aldolase activity, glycerol-3-phosphate dehydrogenase activity, glycerol kinase activity,
Glycerol-Dehydrogenase-Aktivität und Transaldolase-Aktivität, codieren.Glycerol dehydrogenase activity and transaldolase activity.
Fig. 2 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um Teile des Calvin-Benson-Bassham-Zyklus, beteiligt sind; Fructosebisphosphat-Aldolase und Fructose-Bisphosphatase wurden zur Steigerung des Synthesepotentials ersetzt durch Glycerol-3- Phosphat-Dehydrogenase, Glycerol-Kinase, Glycerol-Dehydro- genäse, Fructose-6-Phosphat-Aldolase und Transaldolase.Figure 2 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by parts of the Calvin-Benson-Bassham cycle; Fructose bisphosphate aldolase and fructose bisphosphatase have been replaced by glycerol-3-phosphate dehydrogenase, glycerol kinase, glycerol dehydrogenase, fructose-6-phosphate aldolase and transaldolase to increase their synthetic potential.
(2b) Serin-Zyklus (SC)(2b) serine cycle (SC)
Als Alternative zur Realisierung des CBB steht erfindungsgemäß auch der Serin-Zyklus (SC) zur Verfügung. Aufgrund des hohenAs an alternative to the realization of the CBB, the serine cycle (SC) is also available according to the invention. Due to the high
Energiebedarfs der Fixierung von CO2 im CBB ist mit diesem Weg der CO2-Fixierung eine Produktion von organischen Substanzen (z.B. Lactat) nur möglich, wenn zusätzlich zu CO2 ein weiterer Elektronenakzeptor, beispielsweise Sauerstoff, zur Verfügung steht. Darüber hinaus weist die im CBB bevorzugt eingesetzte Ribulose- bisphosphat-Carboxylase eine vergleichsweise niedrige Aktivität auf. Im SC besteht hingegen vorteilhafterweise die prinzipielle Möglichkeit, auf einen zusätzlichen Elektronenakzeptor neben CO2 gänzlich zu verzichten. Der SC ist nicht angewiesen auf die Fixierung von CO2 in der Reaktion der Ribulosebisphosphat-Carboxylase. Dieses Enzym katalysiert über die CO2-Fixierung hinaus auch eine uner- wünschte Nebenreaktion mit 02 als Substrat, wodurch die Effizienz des Stoffwechselwegs reduziert wird.The energy requirement of fixing CO2 in CBB is a production of organic substances with this path of CO2 fixation (eg lactate) only possible if, in addition to CO2, another electron acceptor, for example oxygen, is available. In addition, the ribulose bisphosphate carboxylase preferably used in CBB has a comparatively low activity. On the other hand, in the SC there is advantageously the possibility in principle to completely dispense with an additional electron acceptor besides CO2. The SC is not dependent on the fixation of CO2 in the reaction of ribulose bisphosphate carboxylase. In addition to CO2 fixation, this enzyme also catalyzes an undesirable side reaction with O 2 as a substrate, which reduces the efficiency of the metabolic pathway.
Die Erfindung sieht in dieser alternativen Variante bevorzugt vor, dass zur CO2-Assimilation überwiegend oder ausschließlich der Serin-Zyklus (SC) realisiert wird. Die erfindungsgemäße Realisierung dieses Stoffwechselwegs sieht primär die Expression einer Formyl- Tetrahydrofolat-Ligase-Aktivität vor.The invention preferably provides in this alternative variant for predominantly or exclusively the serine cycle (SC) to be implemented for CO2 assimilation. The realization of this metabolic pathway according to the invention primarily provides for the expression of a formyl-tetrahydrofolate ligase activity.
Fig. 3 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase, beteiligt sind; zur Assimi- lation des Wasserstoffs und zur Synthese von Glycin werden hier homologe Enzyme verwendet.Fig. 3 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by a formyl tetrahydrofolate ligase; For the assimilation of hydrogen and for the synthesis of glycine, homologous enzymes are used.
Fig. 4 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase, beteiligt sind; zur Syn- these von Glycin werden hier homologe Enzyme verwendet; die Assimilation von Wasserstoff geschieht durch eine rekombinante Hydrogenase. Die erfindungsgemäße Realisierung einer Formyl-Tetrahydrofolat- Ligase-Aktivität stellt vor allem die Einschleusung von Formiat in die Übertragung von C 1 -Körpern sicher, besonders die Übertragung von C1 -Körpern auf Glycin im Zusammenhang mit einer Serin- Hydroxymethyl-Transferase-Aktivität. Dieser Prozess ist vor allem in Verbindung mit der Verwendung von E. coli als Wirtszelle relevant. Bevorzugt ist zusätzlich die homologe oder heterologe Expression einer, bevorzugt cytosolischen, Formiat-Dehydrogenase-Aktivität und/oder einer damit vergleichbaren Aktivität realisiert.Fig. 4 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended to formyl tetrahydrofolate ligase; for the synthesis of glycine, homologous enzymes are used; the assimilation of hydrogen is by a recombinant hydrogenase. The realization according to the invention of a formyl-tetrahydrofolate ligase activity ensures, above all, the introduction of formate into the transfer of C 1 -substances, in particular the transfer of C 1 -substances to glycine in connection with a serine hydroxymethyltransferase activity. This process is particularly relevant in connection with the use of E. coli as a host cell. Preferably, in addition, the homologous or heterologous expression of a, preferably cytosolic, formate dehydrogenase activity and / or a comparable activity is realized.
Vor allem im Zusammenhang mit der Verwendung von E. coli als Wirtszelle im anaeroben Prozess und unter Voraussetzung einer reversiblen Formiat-Hydrogen-Lyase und Energieerzeugung durch eine Protonengradient-erzeugende Formiat-Dehydrogenase-Aktivität reicht die Realisierung der erfindungsgemäßen heterologen Formyl- Tetrahydrofolat-Ligase-Aktivität überraschenderweise aus, um die Einschleusung von Formiat in die Übertragung von C1-Körpem zu sichern. Diese überraschende Erkenntnis macht sich die Erfindung zu nutze, um eine mit geringerem Aufwand transformierte Zelle bereitzustellen. In diesem Zusammenhang werden Reduktionsäquiva- lente, die bei der homologen Formiat-Dehydrogenase-Aktivität anfallen, übertragen auf C1-Tetrahydrofolat, Pyruvat etc. Es wird auf die in Fig. 3 dargestellten erfindungsgemäßen Stoffwechselwege verwiesen.Particularly in connection with the use of E. coli as the host cell in the anaerobic process and assuming a reversible formate hydrogen lyase and energy production by a proton-generating formate dehydrogenase activity, the realization of the heterologous formyl-tetrahydrofolate ligase according to the invention is sufficient. Activity surprisingly to ensure the introduction of formate in the transmission of C1-Körpem. This surprising finding makes use of the invention to provide a transformed cell with less effort. In this connection, reduction equivalents which occur in the case of the homologous formate dehydrogenase activity are transferred to C 1 -tetrahydrofolate, pyruvate, etc. Reference is made to the metabolic pathways according to the invention shown in FIG. 3.
Zur erfindungsgemäßen Realisierung dieses Stoffwechselwegs wird erfindungsgemäß mindestens eine Formyl-Tetrahydrofolat-Ligase- Aktivität exprimiert. Bevorzugt stammt die Formyl-Tetrahydrofolat- Ligase-Aktivität aus dem Mikroorganismus Methylobacterium extor- quens, besonders das Enzym Formyl-Tetrahydrofolat-Ligase ftfL, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 107. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 108 auf.For the realization of this metabolic pathway according to the invention, at least one formyl-tetrahydrofolate ligase activity is expressed according to the invention. The formyl tetrahydrofolate ligase activity preferably originates from the microorganism Methylobacterium extorquens, especially the enzyme formyl tetrahydrofolate ligase ftfL, or is derived from it. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 107. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 108.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, Nucleinsäuremolekül enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenz SEQ ID NO: 107 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequence SEQ ID NO: 107;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenz SEQ ID NO: 108 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules coding for amino acid molecules containing or consisting of the sequence SEQ ID NO: 108; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Aktivität von Formyl-Tetrahydrofolat-Ligase codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of formyl tetrahydrofolate ligase encode.
In einer bevorzugten Variante dieser Ausführung ist der SC unter Einbeziehung einer in der Wirtszelle vorhandenen Glycin-Cleavage- System-Aktivität realisiert. Dies ist vor allem bei Verwendung der Wirtszellen E. coli oder Ralstonia relevant. Erfindungsgemäß bevorzugt wird eine, bevorzugt wirtseigene, Glycin-Cleavage-System- Aktivität exprimiert. Bevorzugt stammt die Glycin-Cleavage-System- Aktivität qcvPHT und Ißd aus E. coli oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist das Glycin-Cleavage- System von E. coli aus 4 Untereinheiten aufgebaut. Bevorzugte, die- se Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 189 für Untereinheit qcvP. SEQ ID NO: 191 für Untereinheit qcvH und SEQ ID NO: 193 für Untereinheit gcvT sowie SEQ ID NO: 63 für Untereinheit lp_d. Das Enzymsystem weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 190, 192, 194 und 64 auf.In a preferred variant of this embodiment, the SC is realized by including a glycine cleavage system activity present in the host cell. This is especially relevant when using the host cells E. coli or Ralstonia. Preferably, according to the invention, a, preferably native, glycine cleavage system activity is expressed. Preferably, the glycine cleavage system activity qcvPHT and Issd is from E. coli or derived therefrom. According to the current state of knowledge, the glycine cleavage system of E. coli is composed of 4 subunits. Preferred, subunit-encoding nucleotide sequences are SEQ ID NO: 189 for subunit qcvP. SEQ ID NO: 191 for subunit qcvH and SEQ ID NO: 193 for subunit gcvT and SEQ ID NO: 63 for subunit lp_d. The enzyme system thus preferably has the amino acid sequences SEQ ID NO: 190, 192, 194 and 64.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, Nucleinsäuremolekül enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which contains at least one nucleic acid molecule, which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 189, 191 , 193, 63 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 189, 191, 193, 63;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 190, 192, 194, 64 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 190, 192, 194, 64; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) be- schriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Aktivität des Glycin-Cleavage-Systems codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an activity of the glycine cleavage Encode system.
In einer bevorzugten alternativen Variante ist vorgesehen, dass, alternativ zum wirtseigenen Glycin-Cleavage-Systems oder gegebenenfalls zusätzlich, die rekombinante Expression von Aktivitäten von Schlüsselenzymen des SC, ausgewählt aus:In a preferred alternative variant, it is provided that, as an alternative to the intrinsic glycine cleavage system or optionally additionally, the recombinant expression of activities of key enzymes of the SC selected from:
Serin-Glyoxylat-Aminotransferase-Aktivität, Hydroxypyruvat-Reduktase-Aktivität, Malat-Thiokinase-Aktivität, Malyl-CoA-Lyase-Aktivität und Isocitrat-Lyase-AktivitätSerine-glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, Malate thiokinase activity, malyl CoA lyase activity and isocitrate lyase activity
und/oder damit vergleichbare Aktivitäten realisiert ist. Unter einer Enzymaktivität eines am SC beteiligten Enzyms wird vorliegend die Fähigkeit zur Umsetzung eines Zwischenprodukts des SC als Substrat in ein anderes Zwischenprodukt des Zyklus als Produkt der Enzymreaktion verstanden. Unter Schlüsselenzymen des SC werden vorliegend Serin-Glyoxylat-Aminotransferase, Hydroxypyruvat- Reduktase, Malat-Thiokinase, Glycerol-Dehydrogenase MaIyI-CoA- Lyase und Isocitrat-Lyase verstanden. Bevorzugt werden alle der vorgenannten Enzymaktivitäten exprimiert, bevorzugt werden alle am SC beteiligten Enzymaktivitäten in ausreichendem Umfang exprimiert. Bevorzugt wird mindestens eine, besonders bevorzugt min- destens zwei dieser Enzymaktivitäten heterolog exprimiert.and / or comparable activities are realized. By an enzyme activity of an enzyme involved in the SC herein is meant the ability to react an intermediate of the SC as a substrate into another intermediate of the cycle as a product of the enzyme reaction. In the present case, key enzymes of the SC are understood as meaning serine-glyoxylate aminotransferase, hydroxypyruvate reductase, malate thiokinase, glycerol dehydrogenase MaIyI-CoA lyase and isocitrate lyase. Preferably, all of the aforementioned enzyme activities are expressed, preferably all enzyme activities involved in SC are expressed to a sufficient extent. Preferably, at least one, more preferably at least two of these enzyme activities are heterologously expressed.
In einer erfindungsgemäß bevorzugten Realisierung der Serin- Glyoxylat-Aminotransferase-Aktivität erlaubt dies die Umgehung der Glycin-Cleavage-System-Aktivität, welche im Zusammenhang mit der Verwendung von E. coli als Wirtszelle vorhanden ist. Sie nutzt gleichzeitig die Aminogruppe aus Serin zur Synthese von Glycin aus Glyoxylat. Die erfindungsgemäß bevorzugte Realisierung einer Hydroxypyruvat-Reduktase-Aktivität erlaubt die Umsetzung des Stoffwechselprodukts der Serin-Glyoxylat-Aminotransferase gemeinsam mit der Glycerat-Kinase im Embden-Meyerhof-Pamas-Weg, welcher in der erfindungsgemäßen Zelle bevorzugt realisiert ist. Die Regeneration von Glyoxylat erfolgt optional, alternativ oder bevorzugt zusätzlich, über eine homologe Phosphoenolpyrovat- Carboxylase-Aktivität, Tricarbonsäure- und Glyoxylat-Weg; dies ist vor allem im Zusammenhang mit der Verwendung von E. coli als Wirtszelle relevant. Die erfindungsgemäße bevorzugte Realisierung der Isocitrat-Lyase-Aktivität wird bevorzugt auch als Teil des Glyoxy- lat-Wegs auch an der Glyoxylat-Regenerierung im Serin-Weg wir- ken. Dies ist vor allem im Zusammenhang mit der Verwendung von E. coli als Wirtszelle relevant.In a preferred embodiment of the serine-glyoxylate aminotransferase activity of the present invention, this allows circumvention of the glycine cleavage system activity present in connection with the use of E. coli as the host cell. It also uses the amino group from serine to synthesize glycine from glyoxylate. The realization of a hydroxypyruvate reductase activity which is preferred according to the invention permits the conversion of the metabolic product of the serine-glyoxylate aminotransferase together with the glycerate kinase in the Embden-Meyerhof-Pamas route, which is preferably realized in the cell according to the invention. The regeneration of glyoxylate is optional, alternatively or preferably in addition, via a homologous phosphoenolpyrovate carboxylase activity, tricarboxylic acid and glyoxylate pathway; This is especially relevant in connection with the use of E. coli as the host cell. The preferred realization of the isocitrate-lyase activity according to the invention is preferably also active as part of the glyoxylate pathway at the glyoxylate regeneration in the serine pathway. This is particularly relevant in connection with the use of E. coli as a host cell.
Bevorzugt stammt die Serin-Glyoxylat-Aminotransferase-Aktivität aus dem Mikroorganismus Methylobacterium extorquens, besonders ist das Enzym Serin-Glyoxylat-Aminotransferase sgaA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidse- quenz ist SEQ ID NO: 113. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 114 auf.Preferably, the serine-glyoxylate aminotransferase activity derives from the microorganism Methylobacterium extorquens, especially the enzyme serine-glyoxylate aminotransferase sgaA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 113. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 114.
Bevorzugt stammt die Hydroxypyruvat-Reduktase-Aktivität aus dem Mikroorganismus Methylobacterium extorquens, besonders ist das Enzym Hydroxypyruvat-Reduktase hprA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 1 15. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 116 auf.The hydroxypyruvate reductase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme hydroxypyruvate reductase is hprA or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 1 15. The enzyme protein therefore preferably has the amino acid sequence SEQ ID NO: 116.
Bevorzugt stammt die Serin-Malat-Thiokinase-Aktivität aus dem Mik- roorganismus Methylobacterium extorquens, besonders ist das Enzym Malat-Thiokinase mtkAB, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist die Malat-Thiokinase aus 2 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 117 für Untereinheit mtkA und SEQ ID NO: 119 für Untereinheit mtkB. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 118 und 120 auf. Bevorzugt stammt die Glycerol-Dehydrogenase Malyl-CoA-Lyase- Aktivität aus dem Mikroorganismus Methylobacterium extorquens, besonders ist das Enzym Glycerol-Dehydrogenase Malyl-CoA-Lyase mcIA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität co- dierende Nucleotidsequenz ist SEQ ID NO: 121 . Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 122 auf.The serine malate thiokinase activity preferably originates from the microorganism Methylobacterium extorquens, in particular the enzyme malate thiokinase is mtkAB, or is derived therefrom. According to the current state of knowledge, the malate thiokinase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 117 for mtkA subunit and SEQ ID NO: 119 for mtkB subunit. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 118 and 120. Preferably, the glycerol dehydrogenase malyl CoA lyase activity is derived from the microorganism Methylobacterium extorquens, especially the enzyme glycerol dehydrogenase malyl CoA lyase mcIA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 121. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 122.
Bevorzugt stammt die Isocitrat-Lyase-Aktivität aus E. coli, besonders ist das Enzym Isocitrat-Lyase aceA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 1 1 1 . Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 1 12 auf. Im Zusammenhang mit der Verwendung einer Wirtszelle vom Stamm E. coli, ist die Expression der Isocitrat- Lyase-Aktivität realisiert durch homologe Expression, bevorzugt durch homologe Überexpression.The isocitrate lyase activity preferably originates from E. coli, in particular the enzyme isocitrate lyase aceA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 11.1. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 1 12. In connection with the use of a host cell of strain E. coli, expression of isocitrate lyase activity is realized by homologous expression, preferably by homologous overexpression.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei, drei, vier, fünf oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five or preferably all nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 1 1 1 , 1 13, 1 15, 1 17, 1 19 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 1 1 1, 1 13, 1 15, 1 17, 1 19;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 1 12, 1 14, 1 16, 1 18, 120 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules coding for amino acid molecules containing or consisting of the sequences SEQ ID NO: 1 12, 1 14, 1 16, 1 18, 120; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) be- schriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt die Aktivität von Schlüsselenzymen des SC, ausgewählt aus: Serin-Glyoxylat-Aminotransferase-Aktivität, Hydroxypyruvat-Reduktase-Aktivität, Malat-Thiokinase- Aktivität, Glycerol-Dehydrogenase Malyl-CoA-Lyase-c) nucleic acid molecules which, with the nucleic acid molecules described under a) and b), are at least 50%, preferably at least 60%, 70%, 80%, particularly preferably have at least 90% or more homology and prefer the activity of key enzymes of the SC selected from: serine-glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, malate-thiokinase activity, glycerol dehydrogenase malyl-CoA-lyase-
Aktivität und Isocitrat-Lyase-Aktivität, codieren.Activity and isocitrate lyase activity.
Fig. 5 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase und die Schlüsselenzyme des Serin-Zyklus (Sehn-Glyoxylat-Aminotransferase, Hydroxypyru- vat-Reduktase, Malat-Thiokinase und Malyl-CoA-Lyase), beteiligt sind; zur Assimilation von Wasserstoff wird eine homologe Hydroge- nase verwendet.5 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly extended by a formyl tetrahydrofolate ligase and the key enzymes of the serine cycle (Sehn-Glyoxylat-Aminotransferase, Hydroxypyrrated Reductase, Malate thiokinase and malyl CoA lyase); For the assimilation of hydrogen a homologous hydrogenase is used.
Fig. 6 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um eine Formyl-Tetrahydrofolat-Ligase und die Schlüsselenzyme des Serin-Zyklus (Serin-Glyoxylat-Aminotransferase, Hydroxypyru- vat-Reduktase, Malat-Thiokinase und Malyl-CoA-Lyase), beteiligt sind; zur Assimilation von Wasserstoff wird eine rekombinante Hydrogenase verwendet.6 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by a formyl tetrahydrofolate ligase and the key enzymes of the serine cycle (serine-glyoxylate aminotransferase, hydroxypyrrated reductase, Malate thiokinase and malyl CoA lyase); For the assimilation of hydrogen, a recombinant hydrogenase is used.
Wird zur erfindungsgemäßen Realisierung einer rekombinanten Hydrogenase-Aktivität das pHG1-Megaplasmid aus Ralstonia eutropha in die erfindungsgemäße Zelle eingeführt, muss zur Unterdrückung des CBB dort zumindest eine Deletion von Genen für Schlüsselenzyme des CBB, welche auf dem kompletten pHG1- Megaplasmid vorhanden sind, erfolgt sein. Bevorzugt sind auf dem Megaplasmid die Gene für Phosphoribulose-Kinase cbbPp (SEQ ID NO: 35) und/oder Ribulosebisphoshat-Carboxylase cbbLp (SEQ ID NO: 37) und cbbSp (SEQ ID NO: 39) deletiert.If the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB , Preferably on the megaplasmid the genes for phosphoribulose kinase cbbPp (SEQ ID NO: 35) and / or ribulose bisphosphate carboxylase cbbLp (SEQ ID NO: 37) and cbbSp (SEQ ID NO: 39).
(2c) reduktiver Tricarbonsäurezyklus (RTCC)(2c) Reductive Tricarboxylic Acid Cycle (RTCC)
Als weitere Alternative zum CBB steht der erfindungsgemäß auch der reduktive Tricarbonsäurezyklus (RTCC) zur Verfügung. Ebenso wie bei der erfindungsgemäßen Variante der Realisierung des SC kann vorteilhafterweise auf einen zusätzlichen Elektronenakzeptor neben CO2 gänzlich verzichtet werden; auf die gegebenenfalls nachteilige Ribulosebisphosphat-Carboxylase-Aktivität des CBB wird in dieser alternativen Weiterbildung ebenfalls verzichtet.As a further alternative to CBB, the invention also provides the reductive tricarboxylic acid cycle (RTCC). As with the variant according to the invention of the realization of the SC, it is advantageously possible to dispense entirely with an additional electron acceptor in addition to CO2; the optionally detrimental ribulose bisphosphate carboxylase activity of the CBB is also omitted in this alternative development.
Unter einer Enzymaktivität eines am RTCC beteiligten Enzyms wird vorliegend die Fähigkeit zur Umsetzung eines Zwischenprodukts des RTCC als Substrat in ein anderes Zwischenprodukt des Zyklus als Produkt der Enzymreaktion verstanden. Unter Schlüsselenzymen des RTCC werden vorliegend Citrat-Lyase, Oxoglutarat- Oxidoreduktase und Fumarat-Reduktase verstanden.By enzyme activity of an enzyme involved in the RTCC herein is meant the ability to react an intermediate of the RTCC as a substrate into another intermediate of the cycle as a product of the enzyme reaction. Key enzymes of the RTCC are understood to mean citrate lyase, oxoglutarate oxidoreductase and fumarate reductase.
Die Erfindung sieht in einer alternativen Variante bevorzugt vor, dass zur CO2-Assimilation überwiegend oder ausschließlich der reduktive Tricarbonsäurezyklus (RTCC) realisiert wird, wobei in der erfin- dungsgemäßen Zelle alle am RTCC beteiligten Enzymaktivitäten in ausreichendem Umfang exprimiert werden. Dazu ist erfindungsgemäß primär vorgesehen, dass die Expression von Aktivitäten von Schlüsselenzymen des RTCC, ausgewählt aus:In an alternative variant, the invention preferably provides for predominantly or exclusively the reductive tricarboxylic acid cycle (RTCC) to be carried out for CO2 assimilation, wherein all the enzyme activities involved in the RTCC are sufficiently expressed in the cell according to the invention. For this purpose, it is primarily provided according to the invention that the expression of activities of key enzymes of the RTCC, selected from:
Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität undCitrate lyase activity, oxoglutarate oxidoreductase activity and
Fumarat-Reduktase-Aktivität und/oder damit vergleichbare Aktivitäten realisiert wird.Fumarate reductase activity and / or comparable activities are realized.
Bevorzugt werden alle der vorgenannten Enzymaktivitäten expri- miert, bevorzugt werden alle am RTCC beteiligten Enzymaktivitäten in ausreichendem Umfang exprimiert. Bevorzugt wird mindestens eine, besonders bevorzugt mindestens zwei dieser Enzymaktivitäten heterolog exprimiert.Preferably, all of the abovementioned enzyme activities are expressed, preferably all enzyme activities involved in the RTCC are expressed to a sufficient extent. Preferably, at least one, more preferably at least two, of these enzyme activities are heterologously expressed.
Bevorzugt stammt die Citrat-Lyase-Aktivität aus dem Mikroorganismus Chlorobium tepidum, besonders ist das Enzym Citrat-Lyase ac- IAB, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Er- kenntnis ist diese Citrat-Lyase aus 2 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 123 für Untereinheit acIA, und SEQ ID NO: 125 für Untereinheit acIB. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 124 und 126 auf.The citrate-lyase activity preferably originates from the microorganism Chlorobium tepidum, especially the enzyme citrate-lyase ac-IAB, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 123 for subunit acIA, and SEQ ID NO: 125 for subunit acIB. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 124 and 126.
Alternativ stammt die Citrat-Lyase-Aktivität aus E. coli, besonders das Enzym Citrat-Lyase citFED, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist diese Citrat-Lyase aus 3 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 127 für Untereinheit citF, SEQ ID NO: 129 für Untereinheit citE und SEQ ID NO: 131 für Untereinheit citD. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 128, 130 und 132 auf. Im Zusammenhang mit der Verwendung einer Wirtszelle vom Stamm E. coli, ist die Expression einer aktiven Citrat-Lyase-Aktivität realisiert durch homologe Expression, bevorzugt durch homologe Überexpression eines, gegebenenfalls durch Spontanmutation, mutierten Allels der ursprünglich inaktiven Citrat-Lyase von E. coli. Bevorzugt stammt die Oxoglutarat-Oxidoreduktase-Aktivität aus dem Mikroorganismus Hydrogenobacter thermophilus, besonders ist das Enzym Oxoglutarat-Oxidoreduktase korAB, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist diese Oxoglutarat- Oxidoreduktase aus 2 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 133 für Untereinheit korA, und SEQ ID NO: 135 für Untereinheit korB. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 134 und 136 auf.Alternatively, the citrate lyase activity is from E. coli, especially the enzyme citrate lyase citFED, or is derived therefrom. According to the current state of knowledge, this citrate lyase is composed of 3 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 127 for subunit citF, SEQ ID NO: 129 for subunit citE, and SEQ ID NO: 131 for subunit citD. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 128, 130 and 132. In connection with the use of a host cell of strain E. coli, the expression of an active citrate lyase activity is realized by homologous expression, preferably by homologous overexpression of, possibly by spontaneous mutation, mutant allele of the originally inactive citrate lyase of E. coli , The oxoglutarate oxidoreductase activity preferably originates from the microorganism Hydrogenobacter thermophilus, in particular the enzyme oxoglutarate oxidoreductase korAB, or is derived therefrom. According to the current state of knowledge, this oxoglutarate oxidoreductase is composed of 2 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 133 for subunit korA, and SEQ ID NO: 135 for subunit korB. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 134 and 136.
Bevorzugt stammt die Fumarat-Reduktase-Aktivität aus E. coli, besonders ist das Enzym Fumarat-Reduktase frdDCBA, oder ist daraus abgeleitet. Nach dem bisherigen Stand der Erkenntnis ist die Fumarat-Reduktase aus 4 Untereinheiten aufgebaut. Bevorzugte, diese Untereinheiten codierenden Nucleotidsequenzen sind SEQ ID NO: 57 für Untereinheit frdD, SEQ ID NO: 55 für Untereinheit frdC, SEQ ID NO: 53 für Untereinheit frdB und SEQ ID NO: 51 für Untereinheit frdA. Das Enzymprotein weist also bevorzugt die Aminosäuresequenzen SEQ ID NO: 58, 56, 54 und 52 auf. Im Zusammenhang mit der Verwendung einer Wirtszelle vom Stamm E. coli, ist die Expres- sion der Fumarat-Reduktase-Aktivität realisiert durch homologe Expression, bevorzugt durch homologe Überexpression.Preferably, the fumarate reductase activity is from E. coli, especially the enzyme fumarate reductase is frdDCBA or derived therefrom. According to the current state of knowledge, the fumarate reductase is composed of 4 subunits. Preferred nucleotide sequences encoding these subunits are SEQ ID NO: 57 for subunit frdD, SEQ ID NO: 55 for subunit frdC, SEQ ID NO: 53 for subunit frdB and SEQ ID NO: 51 for subunit frdA. The enzyme protein therefore preferably has the amino acid sequences SEQ ID NO: 58, 56, 54 and 52. In connection with the use of a host cell of strain E. coli, the expression of fumarate reductase activity is realized by homologous expression, preferably by homologous overexpression.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei, drei, vier, fünf, sechs, sieben, acht, neun, zehn oder bevorzugt alle Nucleinsäuremoleküle enthält, wel- che ausgewählt sind aus der Gruppe bestehend aus: a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 51 , 53, 57, 123, 125, 127, 129, 131 , 133, 135 enthalten o- der daraus bestehen;The invention thus preferably relates to a cell which contains at least one, preferably at least two, three, four, five, six, seven, eight, nine, ten or preferably all nucleic acid molecules which are selected from the group consisting of: a) nucleic acid molecules which contain the sequences SEQ ID NO: 51, 53, 57, 123, 125, 127, 129, 131, 133, 135 or consist thereof;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 52, 54, 56, 58, 124, 126, 128,b) nucleic acid molecules which are suitable for amino acid molecules having the sequences SEQ ID NO: 52, 54, 56, 58, 124, 126, 128,
130, 132, 134, 136 enthalten oder daraus bestehen, codieren; und130, 132, 134, 136 encode or consist of encode; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, be- vorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine Aktivitäten von Schlüsselenzymen des RTCC, ausgewählt aus: Citrat-Lyase-Aktivität, Oxoglutarat- Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität, codieren.c) nucleic acid molecules which have at least 50%, preferably at least 50%, 70%, 80%, more preferably at least 90% or more homology with the nucleic acid molecules described under a) and b) and preferably an activity of key enzymes of the RTCC selected from citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
In einer bevorzugten Variante ist zusätzlich die homologe und bevorzugt die heterologe Expression einer, bevorzugt reversiblen, Pyru- vat:Ferredoxin-Oxidoreduktase-Aktivität beziehungsweise Pyruvat- Synthase-Aktivität und/oder einer damit vergleichbaren Aktivität rea- lisiert. Besonders im Zusammenhang mit der Verwendung von E. coli oder Pseudomonas putida als Wirtszelle ist in einer alternativen Variante vorgesehen, dass durch die homologe oder gegebenenfalls heterologe Expression von Enzymaktivität des Malat-Wegs, bevorzugt durch eine NAD-abhängige Malatenzym-Aktivität, vozugsweise das NAD-abhängige Malatenzym aus E. coli, das Zwischenprodukt Oxalacetat durch Decarboxylierung irreversibel in Pyruvat umgewandelt wird. Alternativ oder zusätzlich ist vorgesehen, das Zwischen- produkt Oxalacetat durch homologe oder gegebenenfalls heterologe Expression von Oxalacetat-Decarboxylase-Aktivität, vorzugsweise die Oxalacetat-Decarboxylase aus E. coli, umzuwandeln. Es ziegt sich, dass vorteilhafterweise sowohl Malatenzym als auch Oxalace- tat-Decarboxylase jeweils allein die erforderliche Funktion der Bereitstellung von Pyruvat aus einem Metaboliten des RTCC (Malat oder Oxalacetat) leisten können.In a preferred variant, in addition, the homologous and preferably the heterologous expression of a, preferably reversible, pyruvate: ferredoxin oxidoreductase activity or pyruvate synthase activity and / or an activity comparable therewith is realized. Particularly in connection with the use of E. coli or Pseudomonas putida as the host cell is provided in an alternative variant, that preferably by the homologous or heterologous expression of enzyme activity of the malate pathway, preferably by an NAD-dependent malate enzyme activity, the NAD Dependent malate enzyme from E. coli, the intermediate oxaloacetate is irreversibly converted into pyruvate by decarboxylation. Alternatively or additionally, the intermediate oxaloacetate by homologous or optionally heterologous expression of oxaloacetate decarboxylase activity, preferably the E. coli oxaloacetate decarboxylase. It will be appreciated that advantageously both malate enzyme and oxaloacetate decarboxylase alone can perform the required function of providing pyruvate from a metabolite of RTCC (malate or oxaloacetate).
Bevorzugt ist die homologe Überexpression mindestens einer der vorgenannten Enzym-Aktivitäten vorgesehen, um vorteilhafterweise zu verhindern, dass Nebenprodukte aus dem Tricarbonsäurezyklus anstelle von Lactat produziert werden. Im Zusammenhang mit der erfindungsgemäßen Herstellung von Lactat/Milchsäure ist daher bevorzugt diese Maßnahme vorgesehen, um den irreversiblen Abfluss aus dem Tricarbonsäurezyklus zu realisieren. In Organismen, die natürlicherweise den RTCC als Fixierungsweg für CO2 einsetzen, ist dieser in der Regel verbunden mit einer Pyruvat: Ferredoxin- Oxidoreduktase-Aktivität. Entgegen der Erwartung führt aber auch der mit Malatenzym-Aktivität oder Oxalacetat-Decarboxylase-Aktivität verbundene Decarboxylierungsschritt, der ebenfalls die Herstellung von Pyruvat aus einem Metaboliten des RTCC zur Folge hat, nicht zu einer Einschränkung der Ersetzbarkeit des RTCC als hocheffizienten Weg zur CO2-Fixierung.Preferably, the homologous overexpression of at least one of the aforementioned enzyme activities is provided to advantageously prevent byproducts from the tricarboxylic acid cycle from being produced instead of lactate. In connection with the production according to the invention of lactate / lactic acid, therefore, this measure is preferably provided in order to realize the irreversible outflow from the tricarboxylic acid cycle. In organisms that naturally use the RTCC as fixation route for CO2, this is usually associated with a pyruvate: ferredoxin oxidoreductase activity. Contrary to expectation, however, the decarboxylation step associated with malate enzyme activity or oxaloacetate decarboxylase activity, which also results in the production of pyruvate from a metabolite of RTCC, does not limit the substitutability of RTCC as a highly efficient pathway for CO2 fixation ,
Fig. 7 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erwei- tert um die Schlüsselenzyme des reduktiven Tricarbonsäurezyklus (Citrat-Lyase, Oxoglutarat-Oxidoreduktase und Fumarat-Reduktase), beteiligt sind. Hierbei wird zur Assimilation von Wasserstoff eine homologe Hydrogenase verwendet. Fig. 8 zeigt die Reaktionsschritte, die an der Umsetzung von CO2 und Wasserstoff in Lactat durch Escherichia coli, rekombinant erweitert um die Schlüsselenzyme des reduktiven Tricarbonsäurezyklus (Citrat-Lyase, Oxoglutarat-Oxidoreduktase und Fumarat-Reduktase), beteiligt sind. Hierbei wird Sauerstoff als terminaler Elektronenakzeptor verwendet. Zur Assimilation von Wasserstoff wird eine rekombi- nante Hydrogenase verwendet.7 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase). In this case, a homologous hydrogenase is used to assimilate hydrogen. Figure 8 shows the reaction steps involved in the conversion of CO2 and hydrogen into lactate by Escherichia coli recombinantly expanded by the key enzymes of the reductive tricarboxylic acid cycle (citrate lyase, oxoglutarate oxidoreductase and fumarate reductase). In this case, oxygen is used as the terminal electron acceptor. For the assimilation of hydrogen, a recombi- nant hydrogenase is used.
Wird zur erfindungsgemäßen Realisierung einer rekombinanten Hydrogenase-Aktivität das pHG1-Megaplasmid aus Ralstonia eutropha in die erfindungsgemäße Zelle eingeführt, muss zur Unterdrückung des CBB dort zumindest eine Deletion von Genen für Schlüsselenzyme des CBB, welche auf dem kompletten pHG1- Megaplasmid vorhanden sind, erfolgt sein. Bevorzugt sind auf dem Megaplasmid die Gene für Phosphoribulose-Kinase cbbPp und/oder Ribulosebisphoshat-Carboxylase cbbLp und cbbSp deletiert.If the pHG1 megaplasmid from Ralstonia eutropha is introduced into the cell according to the invention for the purpose of realizing a recombinant hydrogenase activity according to the invention, at least one deletion of genes for key enzymes of CBB present on the complete pHG1 megaplasmide must have occurred there to suppress CBB , The genes for phosphoribulose kinase cbbPp and / or ribulose bisphosphate carboxylase cbbLp and cbbSp are preferably deleted on the megaplasmid.
(3) Lactat-Svnthese(3) lactate synthesis
Zur Unterstützung des Lactat-Synthesestoffwechsels sieht die Erfindung bevorzugt weiter vor, den potentiellen Abbau von Lactat- Vorläufern zu unterdrücken und den Stofffluss in Richtung Lactat zu lenken. Dies wird bevorzugt realisiert durch Hemmung von gegebenenfalls in der erfindungsgemäßen Zelle vorhandenen Enzymaktivitäten konkurrierender Stoffwechselwege und/oder vergleichbarer Enzymaktivitäten, vorzugsweise durch Hemmung der Expression und/oder Deletion der diese Enzymaktivitäten codierenden Nucleo- tidsequenzen beziehungsweise Gene.In support of the lactate synthesis metabolism, the invention preferably further contemplates suppressing the potential degradation of lactate precursors and directing the material flow toward lactate. This is preferably realized by inhibiting any enzyme activities of competing metabolic pathways and / or comparable enzyme activities present in the cell according to the invention, preferably by inhibiting the expression and / or deletion of the nucleotide sequences or genes coding for these enzyme activities.
Besonders im Zusammenhang mit der Verwendung von E. coli oder Pseudomonas putida als Wirtszelle ist zur Herstellung von Lac- tat/Milchsäure weiter vorgesehen, Acetat-umsetzende Stoffwechselwege zu unterdrücken, um die Bildung von Acetat als unerwünschtem Nebenprodukt zu unterdrücken. Im Zusammenhang mit der Realisierung des CBB wird dazu bevorzugt mindestens ein Enzym, bevorzugt alle Enzyme, ausgewählt aus:Particularly in connection with the use of E. coli or Pseudomonas putida as the host cell, the production of lactic acid further provided to suppress acetate-converting pathways to suppress the formation of acetate as an undesired by-product. In connection with the realization of the CBB is preferably at least one enzyme, preferably all enzymes selected from:
Acetat-Kinase A-/Propionat-Kinase 2-Aktivität Phosphat-Acetyl-Transferase-Aktivität, Phosphoenolpyruvat-Carboxylase-Aktivität, Pyruvat-Formiat-Lyase I-Aktivität, Acetaldehyd-CoA-Dehydrogenase-Aktivität,Acetate Kinase A / Propionate Kinase 2 Activity Phosphate Acetyl Transferase Activity, Phosphoenolpyruvate Carboxylase Activity, Pyruvate Formate Lyase I Activity, Acetaldehyde CoA Dehydrogenase Activity,
Fumarat-Reduktase-Aktivität, Pyruvat-Dehydrogenase-Aktivität, Succinat-Dehydrogenase-AktivitätFumarate reductase activity, pyruvate dehydrogenase activity, succinate dehydrogenase activity
gehemmt beziehungsweise deletiert.inhibited or deleted.
Im Zusammenhang mit der Realisierung des SC oder RTCC wird dazu mindestens ein Enzym, bevorzugt alle Enzyme, ausgewählt aus:In connection with the realization of the SC or RTCC, at least one enzyme, preferably all enzymes, selected from:
Acetat-Kinase A-/Propionat-Kinase 2-Aktivität Phosphat-Acetyl-Transferase-Aktivität, Pyruvat-Formiat-Lyase I-Aktivität,Acetate Kinase A / Propionate Kinase 2 Activity Phosphate Acetyl Transferase Activity, Pyruvate Formate Lyase I Activity,
Acetaldehyd-CoA-Dehydrogenase-Aktivität, Pyruvat-Dehydrogenase-AktivitätAcetaldehyde CoA dehydrogenase activity, pyruvate dehydrogenase activity
gehemmt beziehungsweise deletiert.inhibited or deleted.
Besonders in Verbindung mit der Verwendung einer rekombinanten E. coli Zelle sieht die Erfindung dazu bevorzugt vor: die Hemmung der Phosphat-Acetyltransferase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für p_ta, dargestellt durch die SEQ ID NO: 41 oder codierend für die Aminosäuresequenz SEQ ID NO: 42, und alternativ oder bevor- zugt zusätzlichParticularly in connection with the use of a recombinant E. coli cell, the invention preferably provides for this: the inhibition of the phosphate acetyltransferase activity preferably by deletion of at least one of the genes for p_ta, represented by the SEQ ID NO: 41 or coding for the amino acid sequence SEQ ID NO: 42, and alternatively or preferably in addition
gegebenenfalls (bei CBB) die Hemmung der Phosphoenolpy- ruvat-Carboxylase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für ppc, dargestellt durch die SEQ ID NO: 43 oder codierend für die Aminosäuresequenz SEQ ID NO: 44, wobei die Hemmung der Phosphoenolpyruvat-optionally (in CBB) the inhibition of the phosphoenolpyruvate carboxylase activity, preferably by deletion of at least one of the genes for ppc, represented by SEQ ID NO: 43 or coding for the amino acid sequence SEQ ID NO: 44, wherein the inhibition of phosphoenolpyruvate
Carboxylase-Aktivität ppc in diesem Zusammenhang weniger bevorzugt ist.Carboxylase activity ppc is less preferred in this context.
Besonders in Verbindung mit der Verwendung einer rekombinanten E. coli Zelle im anaeroben Prozess sieht die Erfindung dazu alterna- tiv oder bevorzugt zusätzlich vor:Particularly in connection with the use of a recombinant E. coli cell in the anaerobic process, the invention provides alternatively or preferably additionally:
die Hemmung der Pyruvat-Formiat-Lyase I-Aktivität bevorzugt durch Deletion mindestens eines der Gene für pflB, dargestellt durch die SEQ ID NO: 45 oder codierend für die Aminosäuresequenz SEQ ID NO: 46, und alternativ oder bevorzugt zu- sätzlichthe inhibition of pyruvate formate lyase I activity is preferred by deleting at least one of the genes for pflB, represented by SEQ ID NO: 45 or coding for the amino acid sequence SEQ ID NO: 46, and alternatively or preferably additionally
die Hemmung der Acetat-Kinase A/Propionat-Kinase 2- Aktivität bevorzugt durch Deletion mindestens eines der Gene für ackA, dargestellt durch die SEQ ID NO: 47 oder codierend für die Aminosäuresequenz SEQ ID NO: 48, und alternativ oder bevorzugt zusätzlich die Hemmung der Acetaldehyd-CoA-Dehydrogenase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für ad^ hE, dargestellt durch die SEQ ID NO: 49 oder codierend für die Aminosäuresequenz SEQ ID NO: 50, und alternativ oder bevorzugt zusätzlichthe inhibition of the acetate kinase A / propionate kinase 2 activity is preferred by deleting at least one of the genes for ackA represented by SEQ ID NO: 47 or coding for the amino acid sequence SEQ ID NO: 48, and alternatively or preferably additionally the inhibition of acetaldehyde CoA dehydrogenase activity preferably by deletion of at least one of the genes for ad ^ hE represented by SEQ ID NO: 49 or coding for the amino acid sequence SEQ ID NO: 50, and alternatively or preferably additionally
gegebenenfalls (bei CBB) die Hemmung der anaeroben Fu- marat-Reduktase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für frdABCD, dargestellt durch die SEQ ID NO: 51 , 53, 55 und 57 oder codierend für die Aminosäure- Sequenzen SEQ ID NO: 52, 54, 56 und 58, besonders bevorzugt von frdA und/oder frdB.optionally (in CBB) the inhibition of anaerobic fumarate reductase activity, preferably by deletion of at least one of the genes for frdABCD, represented by SEQ ID NOS: 51, 53, 55 and 57 or coding for the amino acid sequences SEQ ID NO : 52, 54, 56 and 58, more preferably frdA and / or frdB.
Besonders in Verbindung mit der Verwendung einer rekombinanten E. coli Zelle im aeroben Prozess sieht die Erfindung dazu alternativ bevorzugt vor:Particularly in connection with the use of a recombinant E. coli cell in the aerobic process, the invention alternatively provides for this:
die Hemmung der Pyruvat-Dehydrogenase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für aceEF/lpdA, dargestellt durch die SEQ ID NO: 59 und 61 , sowie 63 oder codierend für die Aminosäuresequenzen SEQ ID NO: 60 und 62, und alternativ oder bevorzugt zusätzlichthe inhibition of pyruvate dehydrogenase activity is preferred by deleting at least one of the aceEF / lpdA genes represented by SEQ ID NOs: 59 and 61, 63 or coding for the amino acid sequences SEQ ID NOs: 60 and 62, and alternatively or preferably additionally
die Hemmung der Acetat-Kinase A/Propionat-Kinase 2-the inhibition of acetate kinase A / propionate kinase 2-
Aktivität bevorzugt durch Deletion mindestens eines der Gene für ackA und alternativ oder bevorzugt zusätzlichActivity is preferred by deleting at least one of the genes for ackA and alternatively or preferably additionally
die Hemmung der Acetaldehyd-CoA-Dehydrogenase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für ad; ITE und alternativ oder bevorzugt zusätzlich die Hemmung der Succinat-Dehydrogenase-Aktivität bevorzugt durch Deletion mindestens eines der Gene für sdhABCD, dargestellt durch die SEQ ID NO: 65, 67, 69 und 71 oder codierend für die Aminosäuresequenz SEQ ID NO: 66, 68, 70 und 72, besonders bevorzugt von sdhA und/oder sdhB.the inhibition of acetaldehyde-CoA dehydrogenase activity is preferred by deleting at least one of the genes for ad; ITE and alternatively or preferably in addition the inhibition of succinate dehydrogenase activity, preferably by deletion of at least one of the genes for sdhABCD represented by SEQ ID NO: 65, 67, 69 and 71 or coding for the amino acid sequence SEQ ID NO: 66, 68, 70 and 72, especially preferably from sdhA and / or sdhB.
Zur Herstellung von enantiomerenreinem D-Lactat sieht die Erfindung bevorzugt vor, dass die Expression einer D-Lactat- Dehydrogenase-Aktivität und/oder einer damit vergleichbaren Aktivität realisiert ist. Bevorzugt stammt die D-Lactat-Dehydrogenase- Aktivität aus E. coli, besonders ist es das Enzym Lactat- Dehydrogenase IdhA, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 73. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 74 auf.For the preparation of enantiomerically pure D-lactate, the invention preferably provides that the expression of a D-lactate dehydrogenase activity and / or a comparable activity is realized. Preferably, the D-lactate dehydrogenase activity originates from E. coli, in particular it is the enzyme lactate dehydrogenase IdhA, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 73. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 74.
Alternativ oder bevorzugt zusätzlich stammt die D-Lactat- Dehydrogenase-Aktivität aus Lactobacillus plantarum, besonders ist es das Enzym Lactat-Dehydrogenase IdhD, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 185. Das Enzymprotein weist also bevorzugt die Ami- nosäuresequenz SEQ ID NO: 186 auf.Alternatively or preferably additionally, the D-lactate dehydrogenase activity originates from Lactobacillus plantarum, in particular it is the enzyme lactate dehydrogenase IdhD, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 185. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 186.
Im Zusammenhang mit der Verwendung einer Wirtszelle vom Stamm E. coli, ist die Expression der D-Lactat-Dehydrogenase-Aktivität realisiert durch homologe Expression, bevorzugt durch homologe Überexpression. Im Zusammenhang mit einer aeroben Prozessführung ist die heterologe Expression oder homologe Überexpression einer Lactat-Dehydrogenase-Aktivität obligatorisch. Weiter ist zur Herstellung von enantiomerenreinem D-Lactat bevorzugt vorgesehen, dass die Expression einer D-Lactat-Transporter- Aktivität und/oder einer damit vergleichbaren Aktivität realisiert ist. Bevorzugt stammt die D-Lactat-Transporter-Aktivität aus E. coli, be- sonders ist es der Transporter HdP beziehungsweise IctP, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nuc- leotidsequenz ist SEQ ID NO: 165. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 166 auf.In connection with the use of a host cell of strain E. coli, the expression of D-lactate dehydrogenase activity is realized by homologous expression, preferably by homologous overexpression. In the context of an aerobic process, heterologous expression or homologous overexpression of lactate dehydrogenase activity is mandatory. Furthermore, it is preferably provided for the production of enantiomerically pure D-lactate that the expression of a D-lactate transporter activity and / or a comparable activity is realized. The D-lactate transporter activity preferably originates from E. coli, in particular it is the transporter HdP or IctP, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 165. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 166.
Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, oder bevorzugt alle Nucleinsäuremoleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:The invention thus preferably relates to a cell which contains at least one, or preferably all, nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO: 73, 165, 185 enthalten oder daraus bestehen;a) nucleic acid molecules which contain or consist of the sequences SEQ ID NO: 73, 165, 185;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 74, 166, 186 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 74, 166, 186; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine D-Lactat-Dehydrogenase-Aktivität und/oder D- Lactat-Transporter-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably a D-lactate dehydrogenase activity and / or D-lactate transporter activity.
Vor allem im Zusammenhang mit der Verwendung einer E. coli Zelle zur Herstellung von D-Lactat sieht die Erfindung dazu zusätzlich vor: die Hemmung der homologen L-Lactat-Dehydrogenase- Aktivität, bevorzugt durch Deletion der Gene für HdD, dargestellt durch die SEQ ID NO: 75 oder codierend für die Aminosäuresequenz SEQ ID NO: 76.Especially in connection with the use of an E. coli cell for the production of D-lactate, the invention additionally provides: the inhibition of homologous L-lactate dehydrogenase activity, preferably by deletion of the genes for HdD represented by SEQ ID NO: 75 or coding for the amino acid sequence SEQ ID NO: 76.
Vor allem im Zusammenhang mit der Verwendung einer Lactobacil- lus Zelle zur Herstellung von D-Lactat im anaeroben Prozess sieht die Erfindung dazu zusätzlich vor:Especially in connection with the use of a lactobacillus cell for the production of D-lactate in the anaerobic process, the invention additionally provides:
die Hemmung der homologen L-Lactat-Dehydrogenase- Aktivität, bevorzugt durch Deletion des Gens für IdhLI , darge- stellt durch die SEQ ID NO: 167 oder codierend für die Aminosäuresequenz SEQ ID NO: 168, und alternativ und bevorzugt zusätzlich durch Deletion des Gens für ldhL2, dargestellt durch die SEQ ID NO: 169 oder codierend für die Aminosäuresequenz SEQ ID NO: 170, und alternativ oder bevorzugt zu- sätzlichthe inhibition of the homologous L-lactate dehydrogenase activity, preferably by deletion of the gene for IdhLI, represented by SEQ ID NO: 167 or coding for the amino acid sequence SEQ ID NO: 168, and alternatively and preferably additionally by deletion of the gene for ldhL2 represented by SEQ ID NO: 169 or coding for the amino acid sequence SEQ ID NO: 170, and alternatively or preferably additionally
die Hemmung der homologen Lactat-Racemase-Aktivität, bevorzugt durch Deletion mindestens eines der Gene für Ia1 rABC1 C2E qlpFL dargestellt durch die SEQ ID NO: 173, 175, 177, 179 und 181 oder codierend für die Aminosäurese- quenz SEQ ID NO: 174, 176, 178, 180 und 182.the inhibition of the homologous lactate racemase activity, preferably by deletion of at least one of the genes for Ia 1 rABC1 C2E qlpFL represented by SEQ ID NO: 173, 175, 177, 179 and 181 or coding for the amino acid sequence SEQ ID NO: 174, 176, 178, 180 and 182.
Zur Herstellung von enantiomerenreinem L-Lactat sieht die Erfindung bevorzugt vor, dass die Expression einer L-Lactat- Dehydrogenase-Aktivität und/oder einer damit vergleichbaren Aktivität realisiert ist. Bevorzugt stammt die L-Lactat-Dehydrogenase- Aktivität aus E. coli, besonders ist es das Enzym L-Lactat:Chinon- Oxidoreduktase (L-Lactat-Dehydrogenase) IctD beziehungsweise HdD, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität co- dierende Nucleotidsequenz ist SEQ ID NO: 75. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 76 auf. Im Zusammenhang mit der Verwendung einer Wirtszelle vom Stamm E. coli, ist die homologe Expression der L-Lactat-Dehydrogenase- Aktivität bevorzugt realisiert durch homologe Überexpression der L- LactatChinon-Oxidoreduktase (L-Lactat-Dehydrogenase) IctD beziehungsweise HdD.For the preparation of enantiomerically pure L-lactate, the invention preferably provides that the expression of an L-lactate dehydrogenase activity and / or a comparable activity is realized. The L-lactate dehydrogenase activity preferably originates from E. coli, in particular it is the enzyme L-lactate: quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD, or is derived therefrom. A preferred, this activity co- nucleotide sequence is SEQ ID NO: 75. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 76. In connection with the use of a host cell of strain E. coli, the homologous expression of L-lactate dehydrogenase activity is preferably realized by homologous overexpression of L-lactate-quinone oxidoreductase (L-lactate dehydrogenase) IctD or HdD.
Alternativ oder bevorzugt zusätzlich stammt die L-Lactat- Dehydrogenase-Aktivität aus dem Mikroorganismus Lactobacillus plantarum. Bevorzugt ist es das Enzym Lactat-Dehydrogenase ldhL.1 oder es ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 167. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 168 auf. Alternativ oder zusätzlich ist es das Enzym Lactat-Dehydrogenase ldhL2 oder es ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 169. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 170 auf.Alternatively or preferably additionally, the L-lactate dehydrogenase activity originates from the microorganism Lactobacillus plantarum. It is preferably the enzyme lactate dehydrogenase ldhL.1 or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 167. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 168. Alternatively or additionally, it is the enzyme lactate dehydrogenase ldhL2 or it is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 169. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 170.
Weiter ist zur Herstellung von enantiomerenreinem L-Lactat bevor- zugt vorgesehen, dass die Expression einer L-Lactat-Transporter- Aktivität und/oder einer damit vergleichbaren Aktivität realisiert ist. Bevorzugt stammt die L-Lactat-Transporter-Aktivität aus dem Mikroorganismus Lactobacillus plantarum, besonders ist es der Transporter IctP, oder ist daraus abgeleitet. Eine bevorzugte, diese Aktivität codierende Nucleotidsequenz ist SEQ ID NO: 171. Das Enzymprotein weist also bevorzugt die Aminosäuresequenz SEQ ID NO: 172 auf. Die Erfindung betrifft somit bevorzugt eine Zelle, welche mindestens ein, bevorzugt mindestens zwei, oder bevorzugt alle Nucleinsäure- moleküle enthält, welche ausgewählt sind aus der Gruppe bestehend aus:Furthermore, it is preferably provided for the production of enantiomerically pure L-lactate that the expression of an L-lactate transporter activity and / or a comparable activity is realized. The L-lactate transporter activity preferably originates from the microorganism Lactobacillus plantarum, in particular it is the transporter IctP, or is derived therefrom. A preferred nucleotide sequence encoding this activity is SEQ ID NO: 171. The enzyme protein thus preferably has the amino acid sequence SEQ ID NO: 172. The invention thus preferably relates to a cell which contains at least one, preferably at least two, or preferably all, nucleic acid molecules which are selected from the group consisting of:
a) Nucleinsäuremolekülen, die die Sequenzen SEQ ID NO:a) nucleic acid molecules which have the sequences SEQ ID NO:
167, 171 , 75 enthalten oder daraus bestehen;167, 171, 75 contain or consist of;
b) Nucleinsäuremolekülen, die für Aminosäuremoleküle, die die Sequenzen SEQ ID NO: 168, 172, 76 enthalten oder daraus bestehen, codieren; undb) nucleic acid molecules encoding amino acid molecules containing or consisting of sequences SEQ ID NO: 168, 172, 76; and
c) Nucleinsäuremolekülen, die mit den unter a) und b) beschriebenen Nucleinsäuremolekülen zumindest 50%, bevorzugt zumindest, 60%, 70%, 80%, besonders bevorzugt zumindest 90% oder mehr Homologie aufweisen und bevorzugt eine L-Lactat-Dehydrogenase-Aktivität und/oder ei- ne L-Lactat-Transporter-Aktivität codieren.c) nucleic acid molecules which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology with the nucleic acid molecules described under a) and b), and preferably an L-lactate dehydrogenase activity and / or encode an L-lactate transporter activity.
Vor allem im Zusammenhang mit der Verwendung einer E. coli Zelle zur Herstellung von L-Lactat im anaeroben Prozess sieht die Erfindung dazu zusätzlich vor:Especially in connection with the use of an E. coli cell for the production of L-lactate in the anaerobic process, the invention additionally provides:
die Hemmung der homologen fermentativen D-Lactat- Dehydrogenase-Aktivität, bevorzugt durch Deletion der Gene für IdhA, dargestellt durch die SEQ ID NO: 73 oder codierend für die Aminosäuresequenz SEQ ID NO: 74.the inhibition of the homologous fermentative D-lactate dehydrogenase activity, preferably by deletion of the genes for IdhA, represented by SEQ ID NO: 73 or coding for the amino acid sequence SEQ ID NO: 74.
Vor allem im Zusammenhang mit der Verwendung einer E. coli Zelle zur Herstellung von L-Lactat im aeroben Prozess sieht die Erfindung dazu zusätzlich vor: die Hemmung der homologen D-Lactat-Dehydrogenase- Aktivität, bevorzugt durch Deletion der Gene für d]d, dargestellt durch die SEQ ID NO: 187 oder codierend für die Aminosäuresequenz SEQ ID NO: 188.Especially in connection with the use of an E. coli cell for the production of L-lactate in the aerobic process, the invention additionally provides: the inhibition of the homologous D-lactate dehydrogenase activity, preferably by deletion of the genes for d] d, represented by SEQ ID NO: 187 or coding for the amino acid sequence SEQ ID NO: 188.
Vor allem im Zusammenhang mit der Verwendung einer Lactobacil- lus Zelle zur Herstellung von L-Lactat sieht die Erfindung dazu zusätzlich vor:Especially in connection with the use of a Lactobacillus cell for the production of L-lactate, the invention additionally provides:
die Hemmung der homologen D-Lactat-Dehydrogenase- Aktivität, bevorzugt durch Deletion des Gens für IdhD, darge- stellt durch die SEQ ID NO: 185 oder codierend für die Aminosäuresequenz SEQ ID NO: 186, und alternativ oder bevorzugt zusätzlichthe inhibition of the homologous D-lactate dehydrogenase activity, preferably by deletion of the gene for IdhD, represented by SEQ ID NO: 185 or coding for the amino acid sequence SEQ ID NO: 186, and alternatively or preferably additionally
die Hemmung der homologen Lactat-Racemase-Aktivität, bevorzugt durch Deletion mindestens eines der Gene für Ia1 rABC1 C2E glpFL dargestellt durch die SEQ ID NO: 173,the inhibition of the homologous lactate racemase activity, preferably by deletion of at least one of the genes for Ia 1 rABC1 C2E glpFL represented by SEQ ID NO: 173,
175, 177, 179 und 181 oder codierend für die Aminosäuresequenz SEQ ID NO: 174, 176, 178, 180 und 182.175, 177, 179 and 181 or coding for the amino acid sequence SEQ ID NO: 174, 176, 178, 180 and 182.
Die vorstehend charakterisierten erfindungsgemäßen rekombinanten Mikroorganismen beziehungsweise transgenen biologischen Zellen sind besonders geeignet für die biotechnologische Synthese von Lactat/Milchsäure und anderen Carbonsäuren oder kurzkettigen Fettsäuren. Das sind erfindungsgemäß C2- bis C6- Körper, bevorzugt C3 bis C6-Körper. Bevorzugte Stoffwechselendprodukte, die sich mittels der erfindungsgemäßen Zellen erzeugen lassen, sind kurzkettige Carbonsäuren, vor allem Mono- und Dicarbonsäuren, bevorzugt D-Lactat und L-Lactat, Acetat/Essigsäure, Formi- at/Ameisensäure und Succinat/Bernsteinsäure, sowie ein- und mehrwertige Alkohole, bevorzugt Ethanol. Sie dienen als Ausgangssubstanzen für die weitere organische Synthese, zum Beispiel zur Herstellung von Kunststoffen (z.B. Polylactate). Die erfindungsgemäß herstellbaren Produkte können auch als Energieträger einge- setzt werden, vor allem als Treibstoffe/Kraftstoffe sowie zu deren Herstellung.The above-characterized recombinant microorganisms or transgenic biological cells according to the invention are particularly suitable for the biotechnological synthesis of lactate / lactic acid and other carboxylic acids or short-chain fatty acids. These are according to the invention C2 to C6 body, preferably C3 to C6 body. Preferred metabolic end products which can be produced by means of the cells according to the invention are short-chain carboxylic acids, especially monocarboxylic and dicarboxylic acids, preferably D-lactate and L-lactate, acetate / acetic acid, formic acid / formic acid and succinate / succinic acid, and also mono- and dicarboxylic acids polyhydric alcohols, preferably ethanol. They serve as starting materials for further organic synthesis, for example for the production of plastics (eg polylactates). The products which can be prepared according to the invention can also be used as energy carriers, especially as fuels / fuels and for their production.
Gegenstand der Erfindung ist auch ein Verfahren zur biotechnologischen Herstellung von organischen Kohlenstoffverbindungen, und zwar vor allem C2- bis C6-Körper, als Produkt aus CO2 als Substrat und einem anorganischen Elektronendonor wie Wasserstoff, wobei in Schritt (a) eine erfindungsgemäße Zelle bereitgestellt wird, in Schritt (b) die Zelle mit dem Substrat und dem Elektronendonor in Kontakt gebracht wird und in Schritt (c) die Zelle unter Bedingungen kultiviert wird, unter denen die Substrate umgesetzt und die Zelle die organischen Kohlenstoffverbindundungen bildet. In einem weiteren Schritt (d) wird das Produkt aus dem Kulturmedium und/oder der Zelle isoliert und gegebenenfalls aufgereinigt. Üblicherweise wird das intrazellulär gebildete Produkt aus der Zelle in das Extrazellulärmedium abgegeben.The invention also provides a process for the biotechnological production of organic carbon compounds, especially C2 to C6 bodies, as a product of CO2 as substrate and an inorganic electron donor such as hydrogen, wherein in step (a) a cell according to the invention is provided, in step (b) the cell is contacted with the substrate and the electron donor and in step (c) the cell is cultured under conditions whereby the substrates are reacted and the cell forms the organic carbon bonds. In a further step (d), the product is isolated from the culture medium and / or the cell and optionally purified. Usually, the intracellularly formed product is released from the cell into the extracellular medium.
Die Kultivierung findet bevorzugt in vorzugsweise flüssigem Kulturmedium statt. Es ist bevorzugt vorgesehen, die Zelle dabei unter anaeroben Bedingungen zu kultivieren. Je nach Wirtsorganismus ist es in einer alternativen Variante auch vorgesehen, die Zelle unter aeroben Bedingungen zu kultivieren. Anhand der vorstehenden Be- Schreibung der Erfindung kann der Fachmann die jeweils günstige Enzymausstattung wählen. Bevorzugt ist der Elektronendonor elementarer Wasserstoff, bevorzugt gasförmiger Wasserstoff Bevorzugte alternative Elektronendonoren sind ausgewählt aus Schwefelwasserstoff, elementarem Schwefel, Sulfit, Thiosulfat, Ammonium, Nitrit und Metallen in reduzierter Form.The cultivation preferably takes place in preferably liquid culture medium. It is preferably provided to cultivate the cell under anaerobic conditions. Depending on the host organism, it is also provided in an alternative variant to cultivate the cell under aerobic conditions. On the basis of the above description of the invention, the person skilled in the art can choose the respectively favorable enzyme equipment. The electron donor is preferably elementary hydrogen, preferably gaseous hydrogen. Preferred alternative electron donors are selected from hydrogen sulfide, elemental sulfur, sulfite, thiosulfate, ammonium, nitrite and metals in reduced form.
Als Elektronenakzeptor wird neben CO2, alternativ oder bevorzugt zusätzlich, mindestens eine Verbindung, ausgewählt aus 02, Nitrat, Nitrit, Sulfat, Sulfit, Thiosulfat und Fumarat, verwendet.In addition to CO2, as an electron acceptor, alternatively or preferably additionally, at least one compound selected from O 2, nitrate, nitrite, sulfate, sulfite, thiosulfate and fumarate is used.
Zu dieser Beschreibung gehört ein Sequenzprotokoll; dieses enthält Angaben zu nachstehend aufgelisteten Genen und damit codierten Proteinen:This description includes a sequence listing; this contains information on the genes listed below and proteins encoded with them:
Abkürzung hoxF Bezeichnung NAD-reducing hydrogenase diaphorase moiety large subunit [Ralstonia eutrophaAbbreviation hoxF Name NAD-reducing hydrogenase diaphorase moiety large subunit [Ralstonia eutropha
H16]H16]
EC-Nummer 1 12 1 2EC number 1 12 1 2
NCBI-GI 38637753NCBI-GI 38637753
NCBI-GenelD 2656814NCBI Genel D 2656814
DNA-Sequenz SEQ ID NO 1DNA sequence SEQ ID NO 1
Aminosäure-Sequenz SEQ ID NO 2Amino acid sequence SEQ ID NO 2
Abkürzung hoxU Bezeichnung NAD-reducing hydrogenase diaphorase moiety small subunit [Ralstonia eutrophaAbbreviation hoxU Name NAD-reducing hydrogenase diaphorase moiety small subunit [Ralstonia eutropha
H16]H16]
EC-Nummer 1 12 1 2EC number 1 12 1 2
NCBI-GI 38637754NCBI-GI 38637754
NCBI-GenelD 2656815NCBI Genel D 2656815
DNA-Sequenz SEQ ID NO 3DNA sequence SEQ ID NO 3
Aminosäure-Sequenz SEQ ID NO 4Amino acid sequence SEQ ID NO 4
Abkürzung hoxY Bezeichnung NAD-reducing hydrogenase hydrogenase moiety small subunit [Ralstonia eutrophaAbbreviation hoxY Name NAD-reducing hydrogenase hydrogenase moiety small subunit [Ralstonia eutropha
H16]H16]
EC-Nummer 1 12 1 2EC number 1 12 1 2
NCBI-GI 38637755NCBI-GI 38637755
NCBI-GenelD 2656816 DNA-Sequenz SEQ ID NO 5NCBI Genel D 2656816 DNA sequence SEQ ID NO 5
Aminosäure-Sequenz SEQ ID NO 6Amino acid sequence SEQ ID NO 6
Abkürzung hoxHAbbreviation hoxH
Bezeichnung NAD-reducmg hydrogenase hydrogenase moiety large subunit [Ralstonia eutrophaName NAD-reducmg hydrogenase hydrogenase moiety large subunit [Ralstonia eutropha
H16]H16]
EC-Nummer 1 12 1 2EC number 1 12 1 2
NCBI-GI 38637756NCBI GI 38637756
NCBI-GenelD 2656817NCBI Genel D 2656817
DNA-Sequenz SEQ ID NO 7DNA sequence SEQ ID NO 7
Aminosäure-Sequenz SEQ ID NO 8Amino acid sequence SEQ ID NO 8
Abkürzung hypC1Abbreviation hypC1
Bezeichnung Protein HypC1 involved in metallocenter formation of hydrogenases [Ralstonia eutropha H16]Name of protein HypC1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H16]
EC-NummerEC number
NCBI-GI 38637683NCBI GI 38637683
NCBI-GenelD 2656436NCBI Genel D 2656436
DNA-Sequenz SEQ ID NO 9DNA sequence SEQ ID NO 9
Aminosäure-Sequenz SEQ ID NO 10Amino acid sequence SEQ ID NO 10
Abkürzung hypD1 Bezeichnung Protein HypD1 involved in metallocenter formation of hydrogenases [Ralstonia eutropha H16]Abbreviation hypD1 name protein HypD1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H16]
EC-NummerEC number
NCBI-GI 38637684NCBI GI 38637684
NCBI-GenelD 2656437NCBI Genel D 2656437
DNA-Sequenz SEQ ID NO 11DNA sequence SEQ ID NO 11
Aminosäure-Sequenz SEQ ID NO 12Amino acid sequence SEQ ID NO 12
Abkürzung hypE1 Bezeichnung Protein HypE1 involved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16]Abbreviation hypE1 name protein HypE1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637685NCBI GI 38637685
NCBI-GenelD 2656438NCBI Genel D 2656438
DNA-Sequenz SEQ ID NO 13DNA sequence SEQ ID NO 13
Aminosäure-Sequenz SEQ ID NO 14Amino acid sequence SEQ ID NO 14
Abkürzung hypA1 Bezeichnung Protein HypA1 involved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16] EC-NummerAbbreviation hypA1 Name protein HypA1 involved in metallocene formation of hydrogenases [Ralstonia eutropha H 16] EC number
NCBI-GI 38637680NCBI-GI 38637680
NCBI-GenelD 2656433NCBI Genel D 2656433
DNA-Sequenz SEQ ID NO 15DNA sequence SEQ ID NO 15
Aminosäure-Sequenz SEQ ID NO 16Amino acid sequence SEQ ID NO 16
Abkürzung hypB1 Bezeichnung Protein HypB1 mvolved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16]Abbreviation hypB1 name protein HypB1 mvolved in metallocene formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637681NCBI-GI 38637681
NCBI-GenelD 2656434NCBI Genel D 2656434
DNA-Sequenz SEQ ID NO 17DNA sequence SEQ ID NO 17
Aminosäure-Sequenz SEQ ID NO 18Amino acid sequence SEQ ID NO 18
Abkürzung hypF1 Bezeichnung Protein HypF1 mvolved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16]Abbreviation hypF1 Name protein HypF1 mvolved in metallocene formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637682NCBI-GI 38637682
NCBI-GenelD 2656435NCBI Genel D 2656435
DNA-Sequenz SEQ ID NO 19DNA sequence SEQ ID NO 19
Aminosäure-Sequenz SEQ ID NO 20Amino acid sequence SEQ ID NO 20
Abkürzung hypA2 Bezeichnung Protein HypA2 mvolved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16]Abbreviation hypA2 name protein HypA2 mvolved in metallocene formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637759NCBI-GI 38637759
NCBI-GenelD 2656548NCBI Genel D 2656548
DNA-Sequenz SEQ ID NO 21DNA sequence SEQ ID NO 21
Aminosäure-Sequenz SEQ ID NO 22Amino acid sequence SEQ ID NO 22
Abkürzung hypB2 Bezeichnung Protein HypB2 mvolved in metallocenter formation of hydrogenases [Ralstonia eutropha H 16]Abbreviation hypB2 name protein HypB2 mvolved in metallocene formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637760NCBI GI 38637760
NCBI-GenelD 2656549NCBI Genel D 2656549
DNA-Sequenz SEQ ID NO 23DNA sequence SEQ ID NO 23
Aminosäure-Sequenz SEQ ID NO 24 Abkürzung hypF2 Bezeichnung Protein HypF2 involved in metailocenter formation of hydrogenases [Ralstonia eutropha H 16]Amino acid sequence SEQ ID NO 24 Abbreviation hypF2 name protein HypF2 involved in metailocenter formation of hydrogenases [Ralstonia eutropha H 16]
EC-NummerEC number
NCBI-GI 38637761NCBI GI 38637761
NCBI-GenelD 2656550NCBI Genel D 2656550
DNA-Sequenz SEQ ID NO 25DNA sequence SEQ ID NO 25
Aminosäure-Sequenz SEQ ID NO 26Amino acid sequence SEQ ID NO 26
Abkürzung hoxA Bezeichnung HoxA component of the hydrogen sen- soπng and Signal transduction SystemAbbreviation HoxA Designation HoxA component of the hydrogen transmission and signal transduction system
EC-NummerEC number
NCBI-GI 38637687NCBI-GI 38637687
NCBI-GenelD 2656440NCBI GenelD 2656440
DNA-Sequenz SEQ ID NO 27DNA sequence SEQ ID NO 27
Aminosäure-Sequenz SEQ ID NO 28Amino acid sequence SEQ ID NO 28
Abkürzung hoxB Bezeichnung HoxB component of the hydrogen sen- soπng and Signal transduction systemAbbreviation HoxB Designation HoxB component of the hydrogen transmission and signal transduction system
EC-NummerEC number
NCBI-GI 38637688NCBI-GI 38637688
NCBI-GenelD 2656441NCBI Genel D 2656441
DNA-Sequenz SEQ ID NO 29DNA sequence SEQ ID NO 29
Aminosäure-Sequenz SEQ ID NO 30Amino Acid Sequence SEQ ID NO 30
Abkürzung hoxC Bezeichnung HoxC component of the hydrogen sen- soring and Signal transduction systemAbbreviation HoxC Designation HoxC component of the hydrogen sensing and signal transduction system
EC-NummerEC number
NCBI-GI 38637689NCBI-GI 38637689
NCBI-GenelD 2656442NCBI Genel D 2656442
DNA-Sequenz SEQ ID NO 31DNA sequence SEQ ID NO 31
Aminosäure-Sequenz SEQ ID NO 32Amino acid sequence SEQ ID NO 32
Abkürzung hoxJ Bezeichnung HoxJ histidine protein kinase component of the hydrogen sensoπng and Signal transduction systemAbbreviation hoxJ Designation HoxJ histidine protein kinase component of the hydrogen sensoπng and signal transduction system
EC-NummerEC number
NCBI-GI 38637690NCBI-GI 38637690
NCBI-GenelD 2656443NCBI Genel D 2656443
DNA-Sequenz SEQ ID NO 33DNA sequence SEQ ID NO 33
Aminosäure-Sequenz SEQ ID NO 34 Abkürzung cbbPp Bezeichnung Phosphoπbulokinase [Ralstonia eutrophaAmino acid sequence SEQ ID NO 34 Abbreviation cbbPp Phosphoπbulokinase [Ralstonia eutropha
H16]H16]
EC-Nummer 2 7 1 19EC number 2 7 1 19
NCBI-GI 38638082NCBI-GI 38638082
NCBI-GenelD 2656765NCBI Genel D 2656765
DNA-Sequenz SEQ ID NO 35DNA sequence SEQ ID NO 35
Aminosäure-Sequenz SEQ ID NO 36 Abkürzung cbbLpAmino acid sequence SEQ ID NO 36 Abbreviation cbbLp
Bezeichnung Rιbulose-1 ,5-bιsphosphate carboxy- lase/oxygenase large subunit [Ralstonia eutropha H 16]Description Rιbulose-1, 5-biphosphate carboxy lase / oxygenase large subunit [Ralstonia eutropha H 16]
EC-Nummer 4 1 1 39 NCBI-GI 38638088EC number 4 1 1 39 NCBI-GI 38638088
NCBI-GenelD 2656546 DNA-Sequenz SEQ ID NO 37 Aminosäure-Sequenz SEQ ID NO 38NCBI GenelD 2656546 DNA sequence SEQ ID NO 37 amino acid sequence SEQ ID NO 38
Abkürzung cbbSp Bezeichnung Rιbulose-1 ,5-bιsphosphate carboxy- lase/oxygenase small subunit [Ralstonia eutropha H 16] EC-Nummer 4 1 1 39Abbreviation cbbSp Name Rιbulose-1, 5-biphosphate carboxy lase / oxygenase small subunit [Ralstonia eutropha H 16] EC number 4 1 1 39
NCBI-GI 38638087NCBI-GI 38638087
NCBI-GenelD 2656545NCBI Genel D 2656545
DNA-Sequenz SEQ ID NO 39DNA sequence SEQ ID NO 39
Aminosäure-Sequenz SEQ ID NO 40Amino acid sequence SEQ ID NO 40
Abkürzung ptaAbbreviation pta
Bezeichnung Phosphate acetyltransferase [Escherichia coh K12]Name Phosphate acetyltransferase [Escherichia coh K12]
EC-Nummer 2 3 1 8 NCBI-GI 16130232EC number 2 3 1 8 NCBI-GI 16130232
NCBI-GenelD 946778 DNA-Sequenz SEQ ID NO 41 Aminosäure-Sequenz SEQ ID NO 42 Abkürzung ppcNCBI GenelD 946778 DNA sequence SEQ ID NO 41 amino acid sequence SEQ ID NO 42 Abbreviation ppc
Bezeichnung phosphoenolpyruvate carboxylase [Escherichia coli K12]Name phosphoenolpyruvate carboxylase [Escherichia coli K12]
EC-Nummer 4 1 1 31 NCBI-GI 16131794 NCBI-GenelD 948457EC number 4 1 1 31 NCBI-GI 16131794 NCBI-GenelD 948457
DNA-Sequenz SEQ ID NO 43 Aminosäure-Sequenz SEQ ID NO 44 Abkürzung pflB Bezeichnung pyruvate formate lyase I [Escherichia coliDNA sequence SEQ ID NO 43 amino acid sequence SEQ ID NO 44 Abbreviation pflb Name pyruvate formate lyase I [Escherichia coli
K12]K12]
EC-Nummer 2 3 1 54EC number 2 3 1 54
NCBI-GI 16128870NCBI-GI 16128870
NCBI-GenelD 945514NCBI GenelD 945514
DNA-Sequenz SEQ ID NO 45DNA sequence SEQ ID NO 45
Aminosäure-Sequenz SEQ ID NO 46Amino acid sequence SEQ ID NO 46
Abkürzung ackA Bezeichnung acetate kmase A and propionate kinase 2Abbreviation ackA Designation acetone kmase A and propionate kinase 2
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer 2 7 2 1EC number 2 7 2 1
NCBI-GI 16130231NCBI-GI 16130231
NCBI-GenelD 946775NCBI GenelD 946775
DNA-Sequenz SEQ ID NO 47DNA sequence SEQ ID NO 47
Aminosäure-Sequenz SEQ ID NO 48Amino acid sequence SEQ ID NO 48
Abkürzung adhE Bezeichnung fused acetaldehyde-CoA dehydro- genase/iron-dependent alcohol dehydro- genase/pyruvate-formate lyase deacti- vase [Escherichia coli K12]Abbreviation adhE name fused acetaldehyde-CoA dehydrogenase / iron-dependent alcohol dehydrogenase / pyruvate formate lyase deactivase [Escherichia coli K12]
EC-Nummer 1 2 1 10 / 1 1 1 1EC number 1 2 1 10/1 1 1 1
NCBI-GI 16129202NCBI-GI 16129202
NCBI-GenelD 945837NCBI GenelD 945837
DNA-Sequenz SEQ ID NO 49DNA sequence SEQ ID NO 49
Aminosäure-Sequenz SEQ ID NO 50Amino acid sequence SEQ ID NO 50
Abkürzung frdA Bezeichnung fumarate reductase (anaerobic) catalytic and NAD/flavoprotein subunit [Escherichia coli K12]Abbreviation frdA Name fumarate reductase (anaerobic) catalytic and NAD / flavoprotein subunit [Escherichia coli K12]
EC-Nummer 1 3 99 1EC number 1 3 99 1
NCBI-GI 16131979NCBI-GI 16131979
NCBI-GenelD 948667NCBI GenelD 948667
DNA-Sequenz SEQ ID NO 51DNA sequence SEQ ID NO 51
Aminosäure-Sequenz SEQ ID NO 52Amino acid sequence SEQ ID NO 52
Abkürzung frdB Bezeichnung fumarate reductase (anaerobic), Fe-S subunit [Escherichia coli K12]Abbreviation frdB Name fumarate reductase (anaerobic), Fe-S subunit [Escherichia coli K12]
EC-Nummer 1 3 99 1 NCBI-GI 16131978 NCBI-GenelD 948666 DNA-Sequenz SEQ ID NO 53 Aminosäure-Sequenz SEQ ID NO 54EC No. 1 3 99 1 NCBI-GI 16131978 NCBI-GenelD 948666 DNA sequence SEQ ID NO 53 Amino acid sequence SEQ ID NO 54
Abkürzung frdC Bezeichnung fumarate reductase (anaerobic), mem- brane anchor subunit [Escherichia coliAbbreviation frdC Name fumarate reductase (anaerobic), mem brane anchor subunit [Escherichia coli
K12]K12]
EC-Nummer 1 3 99 1 NCBI-GI 16131977 NCBI-GenelD 948680 DNA-Sequenz SEQ ID NO 55EC No. 1 3 99 1 NCBI-GI 16131977 NCBI-GenelD 948680 DNA sequence SEQ ID NO 55
DNA-Sequenz Aminosäure-Sequenz SEQ ID NO 56DNA sequence amino acid sequence SEQ ID NO 56
Abkürzung frdD Bezeichnung fumarate reductase (anaerobic), mem- brane anchor subunit [Escherichia coliAbbreviation frdD Name fumarate reductase (anaerobic), mem brane anchor subunit [Escherichia coli
K12]K12]
EC-Nummer 1 3 99 1EC number 1 3 99 1
NCBI-GI 16131976NCBI-GI 16131976
NCBI-GenelD 948668NCBI GenelD 948668
DNA-Sequenz SEQ ID NO 57DNA sequence SEQ ID NO 57
Aminosäure-Sequenz SEQ ID NO 58Amino acid sequence SEQ ID NO 58
Abkürzung aceE Bezeichnung pyruvate dehydrogenase, decarboxylase component E1 , thiamin-binding [Escherichia coli K12]Abbreviation aceE name pyruvate dehydrogenase, decarboxylase component E1, thiamine-binding [Escherichia coli K12]
EC-Nummer 1 2 4 1 NCBI-GI 16128107 NCBI-GenelD 944834 DNA-Sequenz SEQ ID NO 59EC No. 1 2 4 1 NCBI-GI 16128107 NCBI-GenelD 944834 DNA sequence SEQ ID NO 59
DNA-Sequenz Aminosäure-Sequenz SEQ ID NO 60DNA sequence amino acid sequence SEQ ID NO 60
Abkürzung aceFAbbreviation aceF
Bezeichnung pyruvate dehydrogenase, dihydrohpoyl- transacetylase component E2 [Escherichia coli K12]Name pyruvate dehydrogenase, dihydrohpoyl transacetylase component E2 [Escherichia coli K12]
EC-Nummer 1 2 4 1 NCBI-GI 16128108EC number 1 2 4 1 NCBI-GI 16128108
NCBI-GenelD 944794 DNA-Sequenz SEQ ID NO 61 Aminosäure-Sequenz SEQ ID NO 62 Abkürzung Ipd (=lpdA)NCBI GenelD 944794 DNA sequence SEQ ID NO 61 amino acid sequence SEQ ID NO 62 abbreviation Ipd (= lpdA)
Bezeichnung hpoamide dehydrogenase, E3 component is part of three enzyme complexes, e g of pyruvate dehydrogenase [Escherichia coli K12]Name hpoamide dehydrogenase, E3 component is part of three enzyme complexes, eg of pyruvate dehydrogenase [Escherichia coli K12]
EC-Nummer 1 8 1 4EC number 1 8 1 4
NCBI-GI 16128109NCBI-GI 16128109
NCBI-GenelD 944854NCBI Genel D 944854
DNA-Sequenz SEQ ID NO 63DNA sequence SEQ ID NO 63
Aminosäure-Sequenz SEQ ID NO 64Amino acid sequence SEQ ID NO 64
Abkürzung sdhA Bezeichnung succinate dehydrogenase, flavoprotein subunit [Escherichia coli K12]Abbreviation sdhA Name succinate dehydrogenase, flavoprotein subunit [Escherichia coli K12]
EC-Nummer 1 3 5 1EC number 1 3 5 1
NCBI-GI 16128698NCBI-GI 16128698
NCBI-GenelD 945402NCBI GenelD 945402
DNA-Sequenz SEQ ID NO 65DNA sequence SEQ ID NO 65
Aminosäure-Sequenz SEQ ID NO 66Amino acid sequence SEQ ID NO 66
Abkürzung sdhB Bezeichnung succinate dehydrogenase, FeS subunitAbbreviation sdhB designation succinate dehydrogenase, FeS subunit
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer 1 3 5 1EC number 1 3 5 1
NCBI-GI 16128699NCBI-GI 16128699
NCBI-GenelD 945300NCBI GenelD 945300
DNA-Sequenz SEQ ID NO 67DNA sequence SEQ ID NO 67
Aminosäure-Sequenz SEQ ID NO 68Amino acid sequence SEQ ID NO 68
Abkürzung sdhC Bezeichnung succinate dehydrogenase, membrane subunit, binds cytochrome b556 [Escherichia coli K12]Abbreviation sdhC name succinate dehydrogenase, membrane subunit, binds cytochrome b556 [Escherichia coli K12]
EC-Nummer 1 3 5 1EC number 1 3 5 1
NCBI-GI 16128696NCBI-GI 16128696
NCBI-GenelD 945316NCBI GenelD 945316
DNA-Sequenz SEQ ID NO 69DNA sequence SEQ ID NO. 69
Aminosäure-Sequenz SEQ ID NO 70Amino acid sequence SEQ ID NO 70
Abkürzung sdhD Bezeichnung succinate dehydrogenase, membrane subunit, binds cytochrome b556 [Escherichia coli K12]Abbreviation sdhD name succinate dehydrogenase, membrane subunit, binds cytochrome b556 [Escherichia coli K12]
EC-Nummer 1 3 5 1 NCBI-GI 16128697 NCBI-GenelD 945322 DNA-Sequenz SEQ ID NO 71EC number 1 3 5 1 NCBI-GI 16128697 NCBI-GenelD 945322 DNA sequence SEQ ID NO 71
DNA-Sequenz Aminosäure-Sequenz SEQ ID NO 72 Abkürzung IdhA Bezeichnung fermentative D-Iactate dehydrogenase,DNA sequence amino acid sequence SEQ ID NO 72 Abbreviation IdhA Name fermentative D-lactate dehydrogenase,
NAD-dependent [Escherichia coli K12]NAD-dependent [Escherichia coli K12]
EC-Nummer 1 1 1 28EC number 1 1 1 28
NCBI-GI 16129341NCBI-GI 16129341
NCBI-GenelD 946315NCBI GenelD 946315
DNA-Sequenz SEQ ID NO 73DNA sequence SEQ ID NO 73
Aminosäure-Sequenz SEQ ID NO 74Amino acid sequence SEQ ID NO 74
Abkürzung IctD (=lldD) Bezeichnung L-Iactate dehydrogenase, FMN-linked [E- schenchia coli K12]Abbreviation IctD (= lldD) Name L-lactate dehydrogenase, FMN-linked [Eschenchia coli K12]
EC-Nummer 1 1 2 3EC number 1 1 2 3
NCBI-GI 16131476NCBI-GI 16131476
NCBI-GenelD 948121NCBI GenelD 948121
DNA-Sequenz SEQ ID NO 75DNA sequence SEQ ID NO. 75
Aminosäure-Sequenz SEQ ID NO 76Amino Acid Sequence SEQ ID NO 76
Abkürzung fbp Bezeichnung fructose-1 ,6-bιsphosphatase I [Escherichia coli K12]Abbreviation fbp name fructose-1, 6-biphosphatase I [Escherichia coli K12]
EC-Nummer 3 1 3 11EC number 3 1 3 11
NCBI-GI 16132054NCBI-GI 16132054
NCBI-GenelD 948753NCBI GenelD 948753
DNA-Sequenz SEQ ID NO 77DNA sequence SEQ ID NO 77
Aminosäure-Sequenz SEQ ID NO 78Amino acid sequence SEQ ID NO. 78
Abkürzung glpX Bezeichnung fructose 1 ,6-bιsphosphatase Il [Escherichia coli K12]Abbreviation glpX Name fructose 1, 6-biphosphatase II [Escherichia coli K12]
EC-Nummer 3 1 3 1 1EC number 3 1 3 1 1
NCBI-GI 16131763NCBI-GI 16131763
NCBI-GenelD 948424NCBI GenelD 948424
DNA-Sequenz SEQ ID NO 79DNA sequence SEQ ID NO 79
Aminosäure-Sequenz SEQ ID NO 80Amino acid sequence SEQ ID NO 80
Abkürzung fbaA Bezeichnung fructose-bisphosphate aldolase, class IlAbbreviation fbaA Name fructose-bisphosphate aldolase, class II
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer 4 1 2 13EC number 4 1 2 13
NCBI-GI 16130826NCBI-GI 16130826
NCBI-GenelD 947415NCBI GenelD 947415
DNA-Sequenz SEQ ID NO 81DNA sequence SEQ ID NO 81
Aminosäure-Sequenz SEQ ID NO 82 Abkürzung fbaB Bezeichnung fructose-bisphosphate aldolase class IAmino Acid Sequence SEQ ID NO 82 Abbreviation fbaB Name fructose-bisphosphate aldolase class I
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer 4 1 2 13 NCBI-GI 90111385EC number 4 1 2 13 NCBI-GI 90111385
NCBI-GenelD 946632 DNA-Sequenz SEQ ID NO 83 Aminosäure-Sequenz SEQ ID NO 84 Abkürzung fsaANCBI-GenelD 946632 DNA sequence SEQ ID NO 83 amino acid sequence SEQ ID NO 84 Abbreviation fsaA
Bezeichnung fructose-6-phosphate aldolase 1 [Escherichia coli K12]Name fructose-6-phosphate aldolase 1 [Escherichia coli K12]
EC-Nummer 4 - - - NCBI-GI 90111174 NCBI-GenelD 945449EC Number 4 - - - NCBI-GI 90111174 NCBI-GenelD 945449
DNA-Sequenz SEQ ID NO 85 Aminosäure-Sequenz SEQ ID NO 86DNA sequence SEQ ID NO. 85 amino acid sequence SEQ ID NO. 86
Abkürzung fsaB Bezeichnung fructose-6-phosphate aldolase 2 [Escherichia coli K12]Abbreviation of the term fructose-6-phosphate aldolase 2 [Escherichia coli K12]
EC-Nummer 4 - . . NCBI-GI 16131784 NCBI-GenelD 948439 DNA-Sequenz SEQ ID NO 87EC number 4 -. , NCBI-GI 16131784 NCBI-GenelD 948439 DNA sequence SEQ ID NO 87
Aminosäure-Sequenz SEQ ID NO 88Amino Acid Sequence SEQ ID NO 88
Abkürzung gpsAAbbreviation gpsA
Bezeichnung glycerol-3-phosphate dehydrogenaseName glycerol-3-phosphate dehydrogenase
(NAD+) [Escherichia coli K12](NAD +) [Escherichia coli K12]
EC-Nummer 1 1 1 94EC number 1 1 1 94
NCBI-GI 16131479NCBI-GI 16131479
NCBI-GenelD 948125NCBI GenelD 948125
DNA-Sequenz SEQ ID NO 89 Aminosäure-Sequenz SEQ ID NO 90DNA sequence SEQ ID NO 89 amino acid sequence SEQ ID NO 90
Abkürzung glpA Bezeichnung sn-glycerol-3-phosphate dehydrogenaseAbbreviation glpA Name sn-glycerol-3-phosphate dehydrogenase
(anaerobic), large subunit, FAD/NAD(P)- binding [Escherichia coli K12](anaerobic), large subunit, FAD / NAD (P) - binding [Escherichia coli K12]
EC-Nummer 1 1 99 5EC number 1 1 99 5
NCBI-GI 16130176NCBI-GI 16130176
NCBI-GenelD 946713NCBI GenelD 946713
DNA-Sequenz SEQ ID NO 91 Aminosäure-Sequenz SEQ ID NO 92 Abkürzung glpB Bezeichnung sn-glycerol-3-phosphate dehydrogenaseDNA sequence SEQ ID NO 91 amino acid sequence SEQ ID NO 92 Abbreviation glpB Name sn-glycerol-3-phosphate dehydrogenase
(anaerobic), membrane anchor subunit(anaerobic), membrane anchor subunit
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer 1 1 99 5EC number 1 1 99 5
NCBI-G! 16130177NCBI-G! 16130177
NCBI-GenelD 946733NCBI GenelD 946733
DNA-Sequenz SEQ ID NO 93DNA sequence SEQ ID NO 93
Aminosäure-Sequenz SEQ ID NO 94Amino Acid Sequence SEQ ID NO 94
Abkürzung glpC Bezeichnung sn-glycerol-3-phosphate dehydrogenaseAbbreviation glpC name sn-glycerol-3-phosphate dehydrogenase
(anaerobic), small subunit [Escherichia coh K12](anaerobic), small subunit [Escherichia coh K12]
EC-Nummer 1 1 99 5EC number 1 1 99 5
NCBI-GI 16130178NCBI-GI 16130178
NCBI-GenelD 946735NCBI GenelD 946735
DNA-Sequenz SEQ ID NO 95DNA sequence SEQ ID NO 95
Aminosäure-Sequenz SEQ ID NO 96Amino Acid Sequence SEQ ID NO 96
Abkürzung glpD Bezeichnung sn-glycerol-3-phosphate dehydrogenase, aerobic, FAD/NAD(P)-bιndιng [Escherichia coli K12]Abbreviation glpD Name sn-glycerol-3-phosphate dehydrogenase, aerobic, FAD / NAD (P) -binding [Escherichia coli K12]
EC-Nummer 1 1 99 5EC number 1 1 99 5
NCBI-GI 16131300NCBI-GI 16131300
NCBI-GenelD 947934NCBI GenelD 947934
DNA-Sequenz SEQ ID NO 97DNA sequence SEQ ID NO 97
Aminosäure-Sequenz SEQ ID NO 98Amino acid sequence SEQ ID NO 98
Abkürzung glpKAbbreviation glpK
Bezeichnung glycerol kinase [Escherichia coli K12]Name glycerol kinase [Escherichia coli K12]
EC-Nummer 2 7 1 30EC number 2 7 1 30
NCBI-GI 16131764NCBI-GI 16131764
NCBI-GenelD 948423NCBI GenelD 948423
DNA-Sequenz SEQ ID NO 99DNA sequence SEQ ID NO 99
Aminosäure-Sequenz SEQ ID NO 100Amino acid sequence SEQ ID NO 100
Abkürzung gldA Bezeichnung glycerol dehydrogenase, NAD [Escherichia coli K12]Abbreviation gldA name glycerol dehydrogenase, NAD [Escherichia coli K12]
EC-Nummer 1 1 1 6EC number 1 1 1 6
NCBI-GI 90111668NCBI-GI 90111668
NCBI-GenelD 948440NCBI GenelD 948440
DNA-Sequenz SEQ ID NO 101DNA sequence SEQ ID NO 101
Aminosäure-Sequenz SEQ ID NO 102 Abkürzung talAAmino acid sequence SEQ ID NO 102 Abbreviation talA
Bezeichnung transaldolase A [Escherichia coli K12]Name transaldolase A [Escherichia coli K12]
EC-Nummer 2 2 1 2EC number 2 2 1 2
NCBI-GI 16130389NCBI-GI 16130389
NCBI-GenelD 947006NCBI GenelD 947006
DNA-Sequenz SEQ ID NO 103DNA sequence SEQ ID NO 103
Aminosäure-Sequenz SEQ ID NO 104Amino acid sequence SEQ ID NO
Abkürzung talBAbbreviation talB
Bezeichnung transaldolase B [Escherichia coli K12]Name transaldolase B [Escherichia coli K12]
EC-Nummer 2 2 1 2EC number 2 2 1 2
NCBI-GI 16128002NCBI-GI 16128002
NCBI-GenelD 944748NCBI GenelD 944748
DNA-Sequenz SEQ ID NO 105DNA sequence SEQ ID NO. 105
Aminosäure-Sequenz SEQ ID NO 106Amino acid sequence SEQ ID NO 106
Abkürzung ftfL Bezeichnung Methylobacteπum extorquens formate- tetrahydrofolate ligase (ftfL)Abbreviation ftfL Designation Methylobacteπum extorquens formate tetrahydrofolate ligase (ftfL)
EC-Nummer 6 3 4 3EC number 6 3 4 3
NCBI-GI 30721807NCBI-GI 30721807
NCBI-GenelDNCBI GenelD
DNA-Sequenz SEQ ID NO 107DNA sequence SEQ ID NO 107
Aminosäure-Sequenz SEQ ID NO 108Amino acid sequence SEQ ID NO. 108
Abkürzung fdhFAbbreviation fdhF
Bezeichnung formate dehydrogenase-H [Escherichia coli K12]Name of formate dehydrogenase-H [Escherichia coli K12]
EC-Nummer 1 2 1 2EC number 1 2 1 2
NCBI-GI 16131905NCBI-GI 16131905
NCBI-GenelD 948584NCBI GenelD 948584
DNA-Sequenz SEQ ID NO 109DNA sequence SEQ ID NO 109
Aminosäure-Sequenz SEQ ID NO 110Amino acid sequence SEQ ID NO 110
Abkürzung aceAAbbreviation aceA
Bezeichnung isocitrate lyase [Escherichia coli K12]Name isocitrate lyase [Escherichia coli K12]
EC-Nummer 4 1 3 1EC number 4 1 3 1
NCBI-GI 16131841NCBI-GI 16131841
NCBI-GenelD 948517NCBI GenelD 948517
DNA-Sequenz SEQ ID NO 111DNA sequence SEQ ID NO 111
Aminosäure-Sequenz SEQ ID NO 112Amino acid sequence SEQ ID NO 112
Abkürzung sgaAAbbreviation of sgaA
Bezeichnung Methylobacteπum extorquens serme- glyoxylate aminotransferaseName Methylobacteπum extorquens serme- glyoxylate aminotransferase
EC-Nummer 2 6 1 45EC number 2 6 1 45
NCBI-GI 439605 (Ausschnitt) NCBI-GenelD DNA-Sequenz SEQ ID NO 113 Aminosäure-Sequenz SEQ ID NO 114NCBI-GI 439605 (detail) NCBI-GenelD DNA sequence SEQ ID NO 113 amino acid sequence SEQ ID NO 114
Abkürzung hprA Bezeichnung Methylobactenum extorquens NADH- dependent hydroxypyruvate reductaseAbbreviation hprA Name Methylobactenum extorquens NADH-dependent hydroxypyruvate reductase
(HPR)(HPR)
EC-Nummer 1 1 1 81 NCBI-GI 439605 (Ausschnitt)EC number 1 1 1 81 NCBI-GI 439605 (detail)
NCBI-GenelD DNA-Sequenz SEQ ID NO 115 Aminosäure-Sequenz SEQ ID NO 116 Abkürzung mtkANCBI-GenelD DNA sequence SEQ ID NO 115 amino acid sequence SEQ ID NO 116 Abbreviation mtkA
Bezeichnung Methylobacteπum extorquens malate thi- okinase (beta subunit)Name Methylobacteπum extorquens malate thiokinase (beta subunit)
EC-Nummer 6 2 1 9 NCBI-GI 505336 (Ausschnitt) NCBI-GenelDEC number 6 2 1 9 NCBI-GI 505336 (detail) NCBI-GenelD
DNA-Sequenz SEQ ID NO 117 Aminosäure-Sequenz SEQ ID NO 118DNA sequence SEQ ID NO 117 amino acid sequence SEQ ID NO 118
Abkürzung mtkB Bezeichnung Methylobacteπum extorquens malate thi- okinase (alpha subunit)Abbreviation mtkB Name Methylobacteπum extorquens malate thiokinase (alpha subunit)
EC-Nummer 6 2 1 9 NCBI-GI 505336 (Ausschnitt) NCBI-GenelD DNA-Sequenz SEQ ID NO 119EC number 6 2 1 9 NCBI-GI 505336 (detail) NCBI-GenelD DNA sequence SEQ ID NO 119
Aminosäure-Sequenz SEQ ID NO 120Amino acid sequence SEQ ID NO 120
Abkürzung mcIAAbbreviation mcIA
Bezeichnung malyl-CoA lyase [Methylobacteπum extorquens]Designation malyl-CoA lyase [Methylobacteπum extorquens]
EC-Nummer 4 1 3 24EC number 4 1 3 24
NCBI-GI 1657785 bzw 28572161 (Ausschnitt)NCBI-GI 1657785 or 28572161 (detail)
NCBI-GenelDNCBI GenelD
DNA-Sequenz SEQ ID NO 121DNA sequence SEQ ID NO 121
Aminosäure-Sequenz SEQ ID NO 122Amino acid sequence SEQ ID NO 122
Abkürzung acIAAbbreviation acIA
Bezeichnung citrate lyase, subunit 2 [Chlorobium tepi dum TLS]Name citrate lyase, subunit 2 [Chlorobium tepi dum TLS]
EC-Nummer 2 3 3 8EC number 2 3 3 8
NCBI-GI 21673914NCBI-GI 21673914
NCBI-GenelD 1006925 DNA-Sequenz SEQ ID NO 123NCBI GenelD 1006925 DNA sequence SEQ ID NO 123
Aminosäure-Sequenz SEQ ID NO 124Amino acid sequence SEQ ID NO 124
Abkürzung aclBAbbreviation aclB
Bezeichnung citrate lyase, subunit 1 [Chlorobium tepi- dum TLS]Name citrate lyase, subunit 1 [Chlorobium tepidum TLS]
EC-Nummer 2 3 3 8EC number 2 3 3 8
NCBI-GI 21673915NCBI-GI 21673915
NCBI-GenelD 1006924NCBI GenelD 1006924
DNA-Sequenz SEQ ID NO 125DNA sequence SEQ ID NO 125
Aminosäure-Sequenz SEQ ID NO 126Amino acid sequence SEQ ID NO 126
Abkürzung citFAbbreviation citF
Bezeichnung citrate lyase, citrate-ACP transferase (alpha) subunit [Escherichia coli K12]Name citrate lyase, citrate-ACP transferase (alpha) subunit [Escherichia coli K12]
EC-Nummer 2 3 3 8EC number 2 3 3 8
NCBI-GI 16128598NCBI-GI 16128598
NCBI-GenelD 945230NCBI GenelD 945230
DNA-Sequenz SEQ ID NO 127DNA sequence SEQ ID NO 127
Aminosäure-Sequenz SEQ ID NO 128Amino acid sequence SEQ ID NO 128
Abkürzung citEAbbreviation citE
Bezeichnung citrate lyase, citryl-ACP lyase (beta) subunit [Escherichia coli K12]Name citrate lyase, citryl-ACP lyase (beta) subunit [Escherichia coli K12]
EC-Nummer 2 3 3 8EC number 2 3 3 8
NCBI-GI 901 11153NCBI-GI 901 11153
NCBI-GenelD 945406NCBI GenelD 945406
DNA-Sequenz SEQ ID NO 129DNA sequence SEQ ID NO 129
Aminosäure-Sequenz SEQ ID NO 130Amino acid sequence SEQ ID NO 130
Abkürzung citDAbbreviation citD
Bezeichnung citrate lyase, acyl carπer (gamma) subunit [Escherichia coli K12]Name citrate lyase, acyl carπer (gamma) subunit [Escherichia coli K12]
EC-Nummer 2 3 3 8EC number 2 3 3 8
NCBI-GI 16128600NCBI-GI 16128600
NCBI-GenelD 945415NCBI GenelD 945415
DNA-Sequenz SEQ ID NO 131DNA sequence SEQ ID NO 131
Aminosäure-Sequenz SEQ ID NO 132Amino acid sequence SEQ ID NO 132
Abkürzung korAAbbreviation korA
Bezeichnung 2-oxoglutarate ferredoxin oxidoreductase alpha subunit [Hydrogenobacter thermo- philus]Name 2-oxoglutarate ferredoxin oxidoreductase alpha subunit [Hydrogenobacter thermophilus]
EC-Nummer -EC number -
NCBI-GI 12583690 (Ausschnitt)NCBI-GI 12583690 (detail)
NCBI-GenelDNCBI GenelD
DNA-Sequenz SEQ ID NO 133 Ammosaure-Sequenz SEQ ID NO 134DNA sequence SEQ ID NO 133 Amino acid sequence SEQ ID NO 134
Abkürzung korBAbbreviation corB
Bezeichnung 2-oxoglutarate ferredoxm oxidoreductase beta subunit [Hydrogenobacter thermo- philus]Name 2-oxoglutarate ferredoxm oxidoreductase beta subunit [Hydrogenobacter thermophilus]
EC-Nummer -EC number -
NCBI-GI 12583690 (Ausschnitt)NCBI-GI 12583690 (detail)
NCBI-GenelDNCBI GenelD
DNA-Sequenz SEQ ID NO 135DNA sequence SEQ ID NO 135
Ammosaure-Sequenz SEQ ID NO 136Amino acid sequence SEQ ID NO 136
Abkürzung hyaAAbbreviation hyaA
Bezeichnung hydrogenase 1 , small subunit [Escherichia coli K12]Name hydrogenase 1, small subunit [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16128938NCBI-GI 16128938
NCBI-GenelD 945579NCBI GenelD 945579
DNA-Sequenz SEQ ID NO 137DNA sequence SEQ ID NO 137
Ammosaure-Sequenz SEQ ID NO 138Amino acid sequence SEQ ID NO 138
Abkürzung hyaBAbbreviation hyaB
Bezeichnung hydrogenase 1 , large subunit [Escherichia coli K12]Name hydrogenase 1, large subunit [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16128939NCBI-GI 16128939
NCBI-GenelD 945580NCBI GenelD 945580
DNA-Sequenz SEQ ID NO 139DNA sequence SEQ ID NO 139
Ammosaure-Sequenz SEQ ID NO 140Amino acid sequence SEQ ID NO 140
Abkürzung hyaCAbbreviation hyaC
Bezeichnung hydrogenase 1 , b-type cytochrome subunit [Escherichia coli K12]Name of hydrogenase 1, b-type cytochrome subunit [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16128940NCBI-GI 16128940
NCBI-GenelD 945581NCBI GenelD 945581
DNA-Sequenz SEQ ID NO 141DNA sequence SEQ ID NO 141
Aminosaure-Seαuenz SEQ ID NO 142 Abkürzung hyaDAmino Acid Sequence SEQ ID NO 142 Abbreviation hyaD
Bezeichnung protein involved in processing of HyaA and HyaB proteins [Escherichia coli K12]HyaA and HyaB proteins [Escherichia coli K12]
EC-Nummer NCBI-GI 16128941 NCBI-GenelD 945575EC number NCBI-GI 16128941 NCBI-GenelD 945575
DNA-Sequenz SEQ ID NO 143 Ammosaure-Sequenz SEQ ID NO 144 Abkürzung hyaE Bezeichnung protein involved in processing of HyaA and HyaB proteins [Escherichia coli K12]DNA sequence SEQ ID NO 143 amino acid sequence SEQ ID NO 144 HyaE abbreviation protein involved in the processing of HyaA and HyaB proteins [Escherichia coli K12]
EC-NummerEC number
NCBI-GI 16128942NCBI-GI 16128942
NCBI-GenelD 945573NCBI GenelD 945573
DNA-Sequenz SEQ ID NO 145DNA sequence SEQ ID NO 145
Aminosäure-Sequenz SEQ ID NO 146Amino acid sequence SEQ ID NO 146
Abkürzung hyaF Bezeichnung protein involved in nickel incorporation ιn- to hydrogenase-1 proteins [Escherichia coli K12]Abbreviation hyaF name protein involved in nickel incorporation ιn- to hydrogenase-1 proteins [Escherichia coli K12]
EC-NummerEC number
NCBI-GI 16128943NCBI-GI 16128943
NCBI-GenelD 945572NCBI GenelD 945572
DNA-Sequenz SEQ ID NO 147DNA sequence SEQ ID NO 147
Aminosäure-Sequenz SEQ ID NO 148Amino acid sequence SEQ ID NO 148
Abkürzung hybOAbbreviation hybO
Bezeichnung hydrogenase 2, small subunit [Escherichia coli K12]Name hydrogenase 2, small subunit [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16130897NCBI-GI 16130897
NCBI-GenelD 945902NCBI GenelD 945902
DNA-Sequenz SEQ ID NO 149DNA sequence SEQ ID NO 149
Aminosäure-Sequenz SEQ ID NO 150Amino acid sequence SEQ ID NO 150
Abkürzung hybAAbbreviation hybA
Bezeichnung hydrogenase 2 4Fe-4S ferredoxin-type component [Escherichia coli K12]Name hydrogenase 2 4Fe-4S ferredoxin-type component [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16130896NCBI-GI 16130896
NCBI-GenelD 944842NCBI GenelD 944842
DNA-Sequenz SEQ ID NO 151DNA sequence SEQ ID NO 151
Aminosäure-Sequenz SEQ ID NO 152Amino acid sequence SEQ ID NO 152
Abkürzung hybBAbbreviation hybB
Bezeichnung hydrogenase 2 cytochrome b type com ponent [Escherichia coli K12]Name hydrogenase 2 cytochrome b type com ponent [Escherichia coli K12]
EC-Nummer 1 12 7 2EC number 1 12 7 2
NCBI-GI 16130895NCBI-GI 16130895
NCBI-GenelD 948615NCBI GenelD 948615
DNA-Sequenz SEQ ID NO 153 Aminosaure-Seαuenz SEQ ID NO 154 Abkürzung hybC Bezeichnung hydrogenase 2, large subunit [Escherichia coli K12]DNA sequence SEQ ID NO 153 amino acid sequence SEQ ID NO 154 Abbreviation hybC Name hydrogenase 2, large subunit [Escherichia coli K12]
EC-Nummer 1 12 7 2 NCBI-GI 16130894EC number 1 12 7 2 NCBI-GI 16130894
NCBI-GenelD 945182 DNA-Sequenz SEQ ID NO 155 Aminosäure-Sequenz SEQ ID NO 156 Abkürzung hybDNCBI GenelD 945182 DNA sequence SEQ ID NO 155 amino acid sequence SEQ ID NO 156 Abbreviation hybD
Bezeichnung maturation element for hydrogenase 2Designation maturation element for hydrogenase 2
[Escherichia coli K12][Escherichia coli K12]
EC-Nummer NCBI-GI 16130893 NCBI-GenelD 948982EC number NCBI-GI 16130893 NCBI-GenelD 948982
DNA-Sequenz SEQ ID NO 157 Aminosäure-Sequenz SEQ ID NO 158DNA sequence SEQ ID NO 157 amino acid sequence SEQ ID NO 158
Abkürzung hybE Bezeichnung hydrogenase 2-specιfιc chaperone [E- scheπchia coli K12]Abbreviation hybE name hydrogenase 2-specιfιc chaperone [Escherichia coli K12]
EC-Nummer NCBI-GI 16130892 NCBI-GenelD 947483 DNA-Sequenz SEQ ID NO 159EC number NCBI-GI 16130892 NCBI-GenelD 947483 DNA sequence SEQ ID NO 159
Aminosäure-Sequenz SEQ ID NO 160Amino acid sequence SEQ ID NO 160
Abkürzung hybF Bezeichnung protein involved with the maturation of hydrogenases 1 and 2 [Escherichia coliAbbreviation hybF Name protein involved with the maturation of hydrogenases 1 and 2 [Escherichia coli
K12]K12]
EC-Nummer NCBI-GI 16130891 NCBI-GenelD 948004 DNA-Sequenz SEQ ID NO 161EC number NCBI-GI 16130891 NCBI-GenelD 948004 DNA sequence SEQ ID NO 161
Aminosäure-Sequenz SEQ ID NO 162Amino acid sequence SEQ ID NO 162
Abkürzung hybGAbbreviation hybG
Bezeichnung hydrogenase 2 accessory protein [Escherichia coli K12]Name of hydrogenase 2 accessory protein [Escherichia coli K12]
EC-NummerEC number
NCBI-GI 16130890NCBI-GI 16130890
NCBI-GenelD 948020NCBI GenelD 948020
DNA-Sequenz SEQ ID NO 163 Aminosäure-Sequenz SEQ ID NO 164 Abkürzung HdP (=lctP)DNA sequence SEQ ID NO 163 amino acid sequence SEQ ID NO 164 Abbreviation HdP (= lctP)
Bezeichnung L-Iactate permease [Escherichia coli K12]Name L-lactate permease [Escherichia coli K12]
EC-Nummer -EC number -
NCBI-GI 16131474NCBI-GI 16131474
NCBI-GenelD 948114NCBI GenelD 948114
DNA-Sequenz SEQ ID NO 165DNA sequence SEQ ID NO 165
Aminosäure-Sequenz SEQ ID NO 166Amino acid sequence SEQ ID NO 166
Abkürzung IdhUAbbreviation IdhU
Bezeichnung L-Iactate dehydrogenase [Lactobacillus plantarum WCFS1]Name L-lactate dehydrogenase [Lactobacillus plantarum WCFS1]
EC-Nummer 1 1 1 27EC number 1 1 1 27
NCBI-GI 28377422NCBI-GI 28377422
NCBI-GenelD 1061886NCBI Genel D 1061886
DNA-Sequenz SEQ ID NO 167DNA sequence SEQ ID NO 167
Aminosäure-Sequenz SEQ ID NO 168Amino acid sequence SEQ ID NO 168
Abkürzung ldhl_2Abbreviation ldhl_2
Bezeichnung L-Iactate dehydrogenase [Lactobacillus plantarum WCFS1]Name L-lactate dehydrogenase [Lactobacillus plantarum WCFS1]
EC-Nummer 1 1 1 27EC number 1 1 1 27
NCBI-GI 28377890NCBI-GI 28377890
NCBI-GenelD 1063343NCBI GenelD 1063343
DNA-Sequenz SEQ ID NO 169DNA sequence SEQ ID NO 169
Aminosaure-Seαuenz SEQ ID NO 170Amino Acid Sequence SEQ ID NO 170
Abkürzung IctP Bezeichnung L-Iactate transport protein [Lactobacillus plantarum WC FS 1]Abbreviation IctP Name L-lactate transport protein [Lactobacillus plantarum WC FS 1]
EC-NummerEC number
NCBI-GI 28378482NCBI-GI 28378482
NCBI-GenelD 1062021NCBI GenelD 1062021
DNA-Sequenz SEQ ID NO 171DNA sequence SEQ ID NO 171
Aminosäure-Sequenz SEQ ID NO 172Amino acid sequence SEQ ID NO 172
Abkürzung larAAbbreviation larA
Bezeichnung lp_0104 [Lactobacillus plantarumDesignation lp_0104 [Lactobacillus plantarum
WCFS1]WCFS1]
EC-Nummer zu 5 1 2 1EC number to 5 1 2 1
NCBI-GI 28377057NCBI-GI 28377057
NCBI-GenelD 1061369NCBI Genel D 1061369
DNA-Sequenz SEQ ID NO 173DNA sequence SEQ ID NO 173
Aminosäure-Sequenz SEQ ID NO 174Amino acid sequence SEQ ID NO 174
Abkürzung larBAbbreviation larB
Bezeichnung Ip 0105 [Lactobacillus plantarumDesignation Ip 0105 [Lactobacillus plantarum
WCFS1] EC-Nummer zu 5 1 2 1WCFS1] EC number to 5 1 2 1
NCBI-GI 28377058NCBI-GI 28377058
NCBI-GenelD 1061366NCBI GenelD 1061366
DNA-Sequenz SEQ ID NO 175DNA sequence SEQ ID NO 175
Aminosäure-Sequenz SEQ ID NO 176Amino acid sequence SEQ ID NO 176
Abkürzung larC1Abbreviation larC1
Bezeichnung lp_0106 [Lactobacillus plantarumDesignation lp_0106 [Lactobacillus plantarum
WCFS1]WCFS1]
EC-Nummer zu 5 1 2 1EC number to 5 1 2 1
NCBI-GINCBI GI
NCBI-GenelD 1061367NCBI GenelD 1061367
DNA-Sequenz SEQ ID NO 177DNA sequence SEQ ID NO 177
Aminosäure-Sequenz SEQ ID NO 178Amino acid sequence SEQ ID NO 178
Abkürzung larC2Abbreviation larC2
Bezeichnung lp_0107 [Lactobacillus plantarumDesignation lp_0107 [Lactobacillus plantarum
WCFS1]WCFS1]
EC-Nummer zu 5 1 2 1EC number to 5 1 2 1
NCBI-GINCBI GI
NCBI-GenelD 1061364NCBI Genel D 1061364
DNA-Sequenz SEQ ID NO 179DNA sequence SEQ ID NO 179
Aminosäure-Sequenz SEQ ID NO 180Amino acid sequence SEQ ID NO 180
Abkürzung glpF1Abbreviation glpF1
Bezeichnung lp_0108, "glycerol uptake facilitator prote in" [Lactobacillus plantarum WCFS1]Description lp_0108, "glycerol uptake facilitator prote in" [Lactobacillus plantarum WCFS1]
EC-Nummer zu 5 1 2 1EC number to 5 1 2 1
NCBI-GI 28377059NCBI-GI 28377059
NCBI-GenelD 1061363NCBI Genel D 1061363
DNA-Sequenz SEQ ID NO 181DNA sequence SEQ ID NO 181
Aminosäure-Sequenz SEQ ID NO 182Amino acid sequence SEQ ID NO 182
Abkürzung larEAbbreviation larE
Bezeichnung lp_0109 [Lactobacillus plantarumDesignation lp_0109 [Lactobacillus plantarum
WCFS1]WCFS1]
EC-Nummer zu 5 1 2 1EC number to 5 1 2 1
NCBI-GI 28377060NCBI-GI 28377060
NCBI-GenelD 1061362NCBI Genel D 1061362
DNA-Sequenz SEQ ID NO 183DNA sequence SEQ ID NO 183
Aminosäure-Sequenz SEQ ID NO 184Amino acid sequence SEQ ID NO 184
Abkürzung IdhDAbbreviation IdhD
Bezeichnung D-Iactate dehydrogenase [Lactobacillus plantarum WCFS1]Name D-lactate dehydrogenase [Lactobacillus plantarum WCFS1]
EC-Nummer 1 1 1 28EC number 1 1 1 28
NCBI-GI 28378688 NCBI-GenelD 1061762NCBI GI 28378688 NCBI Genel D 1061762
DNA-Sequenz SEQ ID NO 185DNA sequence SEQ ID NO 185
Aminosäure-Sequenz SEQ ID NO 186Amino acid sequence SEQ ID NO 186
Abkürzung dldAbbreviation dld
Bezeichnung D-Iactate dehydrogenase, FAD-binding,Name D-lactate dehydrogenase, FAD-binding,
NADH independent [Escherichia coli K12]NADH independent [Escherichia coli K12]
EC-Nummer 1 1 1 28EC number 1 1 1 28
NCBI-GI 16130071NCBI-GI 16130071
NCBI-GenelD 946653NCBI Genel D 946653
DNA-Sequenz SEQ ID NO 187DNA sequence SEQ ID NO 187
Aminosäure-Sequenz SEQ ID NO 188Amino acid sequence SEQ ID NO 188
Abkürzung gcvPAbbreviation gcvP
Bezeichnung glycine decarboxylase, PLP-dependent, subunit (protein P) of glycine cleavage complex [Escherichia coli K12]Name glycine decarboxylase, PLP-dependent, subunit (protein P) of glycine cleavage complex [Escherichia coli K12]
EC-Nummer 1 4 4 2EC number 1 4 4 2
NCBI-GI 16130805NCBI-GI 16130805
NCBI-GenelD 947394NCBI GenelD 947394
DNA-Sequenz SEQ ID NO 189DNA sequence SEQ ID NO 189
Aminosäure-Sequenz SEQ ID NO 190Amino acid sequence SEQ ID NO 190
Abkürzung gcvHAbbreviation gcvH
Bezeichnung glycine cleavage complex lipoylproteinDesignation glycine cleavage complex lipoylprotein
[Escherichia coli K12][Escherichia coli K12]
EC-NummerEC number
NCBI-GINCBI GI
NCBI-GenelDNCBI GenelD
DNA-Sequenz SEQ ID NO 191DNA sequence SEQ ID NO 191
Aminosäure-Sequenz SEQ ID NO 192Amino acid sequence SEQ ID NO 192
Abkürzung gcvT Bezeichnung aminomethyltransferase, tetrahydrofolate- dependent, subunit (T protein) of glycine cleavage complex [Escherichia coli K12]Abbreviation gcvT Name aminomethyltransferase, tetrahydrofolate-dependent, subunit (T protein) of glycine cleavage complex [Escherichia coli K12]
EC-Nummer 2 1 2 10EC number 2 1 2 10
NCBI-GI 16130807NCBI-GI 16130807
NCBI-GenelD 947390NCBI GenelD 947390
DNA-Sequenz SEQ ID NO 193DNA sequence SEQ ID NO 193
Aminosäure-Sequenz SEQ ID NO 194Amino acid sequence SEQ ID NO 194
Abkürzung Ipd (= IpdA) Bezeichnung hpoamide dehydrogenase, ιs part of three enzyme complexes [Escherichia coli K12]Abbreviation Ipd (= IpdA) Name hpoamide dehydrogenase, part of three enzyme complexes [Escherichia coli K12]
EC-Nummer 1 8 1 4 NCBI-GI 16128109 NCBI-GenelD 944854EC number 1 8 1 4 NCBI-GI 16128109 NCBI Genel D 944854
DNA-Sequenz SEQ ID NO 195DNA sequence SEQ ID NO 195
Aminosäure-Sequenz SEQ ID NO 196Amino acid sequence SEQ ID NO 196
Ausführungsbeispiel: Synthese von Milchsäure aus CO2 und H2 in einem rekombinanten E. coli-StammExemplary embodiment: Synthesis of lactic acid from CO2 and H2 in a recombinant E. coli strain
Ausgangspunkt für die Transformation ist der E. coli-Stamm K12. Zur Realisierung der Hydrogenase-Aktivität, des Serin-Zyklus und der Lactat-Synthese wurden Expressionskassetten für: NAD- reduzierende cytosolische Hydrogenase-Aktivität aus Ralstonia , Strukturgene hoxFUYH und Reifungsgene hvpC1 , hypD1 , hypE1 und hypABF, sowie Formyl-Tetrahydrofolat-Ligase-Aktivität aus Me- thylobacterium, speziell enthaltend Nucleinsäuremoleküle mit den Sequenzen SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19 und 107, in Expressionsvektoren pUC oder pPCU18 oder ähnlichen clo- niert/ligiert. Die Ligation der Plasmid-DNA erfolgt in an sich bekannter Weise. Beispielsweise wird der Vektor durch Hydrolyse mit Restriktionsendonuclease geöffnet und die entsprechenden DNA- Sequenzen inseriert. Die Ligation erfolgt beispielsweise durch Kas- settenmutagenese.The starting point for the transformation is E. coli strain K12. To realize the hydrogenase activity, the serine cycle and the lactate synthesis, expression cassettes were used for: NAL-reducing cytosolic hydrogenase activity from Ralstonia, structural hoxFUYH and maturation genes hvpC1, hypD1, hypE1 and hypABF, as well as formyl-tetrahydrofolate ligase activity from mammothacterium, especially containing nucleic acid molecules having the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 107, in expression vectors pUC or pPCU18 or the like cloned / ligated. The ligation of the plasmid DNA is carried out in a conventional manner. For example, the vector is opened by hydrolysis with restriction endonuclease and the corresponding DNA sequences are inserted. Ligation takes place, for example, by cassette mutagenesis.
Die Transformation transformationskompetenter E. coli-Zellen sowie die Herstellung transformationskompetenter E. coli-Zellen erfolgt vorzugsweise nach dem Protokoll von Hanahan, 1985 (Hanahan, D. in Glover, D.M. (ed.) DNA Cloning: „A Practical Approach" IRL Press Oxford 109-135). Zur Herstellung der transformationskompetenten E. coli-Zellen werden E. coli K12-Stämme, beispielsweise TH1 , TH2, TH5, TH5α, C600, Top 10, XL1-Blue und deren Derivate eingesetzt. Aus einer Vorabkultur aus einer Dauerkultur wird eine frische Zellkolonie in 5 ml SOB-Medium suspendiert und unter Schütteln bei 37°C bis zu einer Zelldichte von 1 bis 2 x 108/ml kultiviert. Anschließend wird 1 :1 mit 40% Glycerol/60 % SOB-Medium (1 % Hefeextrakt, 2 % Bacto- Trypton-mmol/l NaCI, 2,5 mmol/l KCl, nach Autoklavieren auf 10 mmol/l MgCI2 und MgSO4 einstellen) verdünnt und auf Eis gekühlt. Aus der Vorabkultur wird ein Zellausstrich auf eine LM-Platte angelegt und über Nacht bei 37°C inkubiert. Daraus werden jeweils 5 Kolonien mit einem Durchmesser von etwa 0,5 bis 1 mm abgeimpft und in 50 ml SOB-Medium angeimpft; die Zellen werden für etwa 3 Stunden bis zu einer Dichte von 4 bis 7 x 107/ml bei 37°C unter Schütteln kultiviert. Die Zellen werden auf Eis gekühlt und in Zentrifugenröhr- chen überführt und bei 2500 rpm bei 4°C für 5 Minuten sedimentiert. Das Sediment wird mit 17 ml LM-Agar-FSB-Puffer resuspendiert und in Eis inkubiert. Der Vorgang wird gegebenenfalls wiederholt. Schließlich wird das Sediment in 5 ml LM-Agar-FSB-Puffer re- suspendiert und 5 Minuten in Eis inkubiert. Dann werden 140 μl DMSO zugegeben, 5 Minuten in Eis inkubiert, nochmals 140 μl DMSO zugegeben, und 5 Minuten in Eis inkubiert. Die Zellsuspension kann beispielsweise in 1 ,5 ml-Reaktionsgefäßen nach langsamen Tieffrieren bei -700C mehrere Monate aufbewahrt werden.The transformation of transformation-competent E. coli cells and the production of transformation-competent E. coli cells is preferably carried out according to the protocol of Hanahan, 1985 (Hanahan, D. in Glover, DM (ed.)) DNA Cloning: "A Practical Approach" IRL Press Oxford 109-135). To prepare the transformation-competent E. coli cells, E. coli K12 strains, for example TH1, TH2, TH5, TH5α, C600, Top 10, XL1-Blue and their derivatives are used. From a preliminary culture from a permanent culture, a fresh cell colony is suspended in 5 ml SOB medium and cultured with shaking at 37 ° C to a cell density of 1 to 2 x 10 8 / ml. Subsequently, 1: 1 with 40% glycerol / 60% SOB medium (1% yeast extract, 2% Bacto-trypton-mmol / l NaCl, 2.5 mmol / l KCl, after autoclaving to 10 mmol / l MgCl 2 and MgSO 4 ) diluted and cooled on ice. From the pre-culture, a cell smear is applied to an LM plate and incubated overnight at 37 ° C. From each of 5 colonies are inoculated with a diameter of about 0.5 to 1 mm and inoculated in 50 ml of SOB medium; the cells are cultured for about 3 hours to a density of 4 to 7 x 10 7 / ml at 37 ° C with shaking. The cells are cooled on ice and transferred to centrifuge tubes and sedimented at 2500 rpm at 4 ° C for 5 minutes. The sediment is resuspended with 17 ml LM agar FSB buffer and incubated in ice. The process is repeated if necessary. Finally, the sediment is resuspended in 5 ml LM agar FSB buffer and incubated for 5 minutes in ice. Then 140 ul DMSO are added, incubated for 5 minutes in ice, added again 140 ul DMSO, and incubated for 5 minutes in ice. The cell suspension may, for example in 1, 5 ml reaction vessels after slow deep-freezing are stored at -70 0 C for several months.
Die Ligation wird in einem molaren Verhältnis von DNA zu Vektor- DNA von 1 :1 bis 1 :3 durchgeführt. Der Ligationsansatz: 4 μl steriles Wasser, 2 μl 5 X Ligasepuffer (250 mmol/l Tris-HCI, pH 7,6, 50 mmol/l MgCI2, 5 mmol/l ATP, 5 mmol/l DTT, 25 % PEE (w/v)), 1 ml amplifizierte DNA, 2 μl Vektor-DNA (mit 3'-T-Überhang in 1 μl T4- Ligase, wird in einem 1 ,5 ml-Reaktionsgefäß vorgelegt und nach Vermischen über Nacht bei 14°C inkubiert. Jeweils 1 - 5 μl des Ansatzes werden zur Transformation von transformationskompetenten E. coli-Zellen eingesetzt; gegebenenfalls kann der Ligationsansatz bei -200C gelagert werden.The ligation is carried out in a molar ratio of DNA to vector DNA of 1: 1 to 1: 3. The ligation mixture: 4 .mu.l of sterile water, 2 .mu.l of 5 × Ligasebuffer (250 mmol / l Tris-HCl, pH 7.6, 50 mmol / l MgCl 2 , 5 mmol / l ATP, 5 mmol / l DTT, 25% PEE ( w / v)), 1 ml of amplified DNA, 2 μl of vector DNA (with 3 'T overhang in 1 μl T4). Ligase, is placed in a 1, 5 ml reaction vessel and incubated after mixing overnight at 14 ° C. In each case 1-5 μl of the mixture are used to transform transformation-competent E. coli cells; where appropriate, the ligation mixture may be stored at -20 0 C.
Zur Transformation der kompetenten E. coli-Zellen werden jeweils 30 μl der zu tranformierenden DNA vorgelegt und bei Kühlung in Eis je 200 μl der Zellsuspension zugegeben und etwa 30 Minuten auf Eis inkubiert. Anschließend wird für 90 Minuten bei 42°C ohne Schütteln inkubiert und danach für 2 Minuten auf Eis abgekühlt. Nach Zugabe von 800 μl SOC-Medium (1 % Hefeextrakt, 2 % Bacto-Trypton- mmol/l NaCI, 2,5 mmol/l KCl, nach Autoklavieren auf 10 mmol/l MgCI2 und MgSO4 einstellen und zusätzlich auf 20 mmol/l Glucolse einstellen) wird bei 37°C bei etwa 200 RPM für etwa 1 Stunde inku- biert. Je 200 μl einer geeigneten Verdünnung des Transformationsansatzes wird auf LB-AMP-Platten ausplattiert. Die Inkubation erfolgt bei 37°C über Nacht.In order to transform the competent E. coli cells, in each case 30 .mu.l of the DNA to be transformed are introduced and, when cooled in ice, 200 .mu.l of the cell suspension are added and incubated on ice for about 30 minutes. The mixture is then incubated for 90 minutes at 42 ° C without shaking and then cooled for 2 minutes on ice. After addition of 800 .mu.l SOC medium (1% yeast extract, 2% Bacto-tryptone mmol / l NaCl, 2.5 mmol / l KCl, after autoclaving to 10 mmol / l MgCl 2 and MgSO 4 and in addition to 20 mmol / l adjust glucolose) is incubated at 37 ° C at about 200 RPM for about 1 hour. 200 μl each of a suitable dilution of the transformation mixture is plated out on LB-AMP plates. Incubation is at 37 ° C overnight.
Zur Selektion der rekombinanten Zellen werden Selektionsplatten verwendet, wobei jeweils 100 μl einer Mischung aus 10 μl 100 mmol/l IPTG, 40 μl 3 % X-GaI und 50 μl SOC-Medium auf einer LB- AMP-Agarplatte ausplattiert werden und diese für etwa 1 Stunde bei 37°C inkubiert werden. Nach Inkubation werden jeweils 200 μl der Zellsuspension auf der Selektionsplatte ausplattiert. Anhand des α- Komplementationstests lassen sich die Kolonien, die kein rekombi- nantes Plasmid enthalten, aufgrund der Blaufärbung von den rekombinanten Clonen, welche keine Färbung aufweisen, leicht unterscheiden. Die stabile heterlologe Expression in den so erhältlichen rekombinanten E. coli Zellen wird in an sich bekannter Weise verifiziert.To select the recombinant cells selection plates are used, in each case 100 .mu.l of a mixture of 10 .mu.l 100 mmol / l IPTG, 40 .mu.l 3% X-GaI and 50 .mu.l SOC medium are plated on an LB-AMP agar plate and this for about Incubated for 1 hour at 37 ° C. After incubation, in each case 200 μl of the cell suspension are plated out on the selection plate. On the basis of the α-complementation test, the colonies which do not contain a recombinant plasmid can easily be distinguished from the recombinant clones which have no coloration due to the blue coloration. The stable heterologous expression in the so available Recombinant E. coli cells are verified in a conventional manner.
Die erhaltenen rekombinanten E. coli Zellen werden nach Inokulation in einem 50 L-Fermenter mit Kulturmedium angesetzt und bei 25 bis 40 0C kultiviert. Die Zugabe des C3 - C6-Substrates, hier Glucose oder alternative Glycerol, erfolgt in das Kulturmedium.The resulting recombinant E. coli cells are prepared after inoculation in a 50 L fermenter with culture medium and cultured at 25 to 40 0 C. The addition of the C3 - C6 substrate, here glucose or alternative glycerol, takes place in the culture medium.
In verschiedenen Ansätzen wird die Kultur unter Einleitung von gasförmigem CO2 und H2 im Reaktor kultiviert. In various approaches, the culture is cultured with introduction of gaseous CO2 and H2 in the reactor.

Claims

Patentansprüche claims
1. Transgene biologische Zelle, enthaltend in exprimierbarer Form:1. Transgenic biological cell containing in expressible form:
(1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; und(1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Enzymaktivität eines Kohlenstoff fixierenden Stoffwechselwegs, und zwar ausgewählt aus:(2) at least one heterologous nucleic acid molecule encoding enzyme activity of a carbon fixing pathway selected from:
- Formyl-Tetrahydrofolat-Ligase-Aktivität; undFormyl-tetrahydrofolate ligase activity; and
- Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität.Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
2. Zelle nach Anspruch 1 , enthaltend :2. Cell according to claim 1, comprising:
(3) mindestens ein heterologes Nucleinsäuremolekül mit einer Nucleotidsequenz, welche für mindestens eine Enzymaktivität, ausgewählt aus:(3) at least one heterologous nucleic acid molecule having a nucleotide sequence which is responsible for at least one enzyme activity selected from:
- Lactat-Dehydrogenase;Lactate dehydrogenase;
- Lactat-Oxidoreduktase;- lactate oxidoreductase;
- Enzymaktivität des Methylglyoxalwegs; und- enzyme activity of the methylglyoxal pathway; and
- Aldehyd-Dehydrogenase - Aldehyde dehydrogenase
3. Zelle nach Anspruch 1 oder 2, enthaltend :3. Cell according to claim 1 or 2, comprising:
heterologes Nucleinsäuremolekül, codierend für Formyl- Tetrahydrofolat-Ligase-Aktivität, und zusätzlich mindestens ein Nucleinsäuremolekül, codierend für Enzymaktivität des Glycin- Cleavage-Systems.heterologous nucleic acid molecule encoding formyl-tetrahydrofolate ligase activity, and additionally at least one nucleic acid molecule encoding enzyme activity of the glycine cleavage system.
4. Zelle nach Anspruch 1 oder 2, enthaltend :4. Cell according to claim 1 or 2, comprising:
heterologes Nucleinsäuremolekül, codierend für Formyl- Tetrahydrofolat-Ligase-Aktivität, und zusätzlich mindestens ein Nucleinsäuremolekül, codierend für Enzymaktivität, ausgewählt aus:heterologous nucleic acid molecule encoding formyl-tetrahydrofolate ligase activity, and additionally at least one nucleic acid molecule encoding enzyme activity selected from:
Serin-Glyoxylat-Aminotransferase-Aktivität, Hydroxypyruvat-Reduktase-Aktivität, Malat-Thiokinase-Aktivität, Malyl-CoA-Lyase-Aktivität und Isocitrat-Lyase-Aktivität.Serine glyoxylate aminotransferase activity, hydroxypyruvate reductase activity, malate thiokinase activity, malyl CoA lyase activity and isocitrate lyase activity.
5. Zelle nach einem der vorstehenden Ansprüche, enthaltend zusätzlich mindestens ein Nucleinsäuremolekül, codierend für For- miat-Dehydrogenase-Aktivität.5. A cell according to any one of the preceding claims additionally containing at least one nucleic acid molecule encoding formate dehydrogenase activity.
6. Zelle nach einem der vorstehenden Ansprüche, enthaltend zusätzlich mindestens ein Nucleinsäuremolekül, codierend für Lac- tat-Dehydrogenase-Aktivität. 6. A cell according to any one of the preceding claims additionally containing at least one nucleic acid molecule encoding lactate dehydrogenase activity.
7. Zelle nach einem der vorstehenden Ansprüche, wobei das mindestens eine Nucleinsäuremolekül, codierend für Hydrogenase- Aktivität, ausgewählt ist aus:A cell according to any one of the preceding claims wherein the at least one nucleic acid molecule encoding hydrogenase activity is selected from:
- membranständiger Hydrogenase-Aktivität aus dem Mikroor- ganismus E. coli, ausgewählt aus Hydrogenase hyaABC und- membrane-bound hydrogenase activity from the microorganism E. coli, selected from hydrogenase hyaABC and
Hydrogenase hybOCAB; undHydrogenase hybOCAB; and
- cytoplasmatischer NAD-reduzierender Hydrogenase-Aktivität aus dem Mikroorganismus Ralstonia eutropha mit den Strukturgenen hoxFUYH.Cytoplasmic NAD-reducing hydrogenase activity from the microorganism Ralstonia eutropha with the structural genes hoxFUYH.
8. Zelle nach einem der vorstehenden Ansprüche, ausgewählt aus: Bakterien der Gattungen Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Lactobacillus, Lacto- coccus und synthetischen Mikroorganismen, Membranvesikeln, Membrankompartimenten und Teilen oder Fragmenten davon.8. Cell according to one of the preceding claims, selected from: bacteria of the genera Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Lactobacillus, Lactococcus and synthetic microorganisms, membrane vesicles, membrane compartments and parts or fragments thereof.
9. Expressionskassette zur Transformation einer Wirtszelle, enthaltend:9. An expression cassette for transforming a host cell, comprising:
(1 ) mindestens ein Nucleinsäuremolekül, codierend für Hydrogenase-Aktivität; und(1) at least one nucleic acid molecule encoding hydrogenase activity; and
(2) mindestens ein heterologes Nucleinsäuremolekül, codierend für Enzymaktivität ausgewählt aus:(2) at least one heterologous nucleic acid molecule encoding enzyme activity selected from:
- Formyl-Tetrahydrofolat-Ligase-Aktivität;Formyl-tetrahydrofolate ligase activity;
- Citrat-Lyase-Aktivität, Oxoglutarat-Oxidoreduktase-Aktivität und Fumarat-Reduktase-Aktivität. Citrate lyase activity, oxoglutarate oxidoreductase activity and fumarate reductase activity.
10. Vektor, enthaltend die Expressionskassette nach Anspruch 9, der geeignet ist, die Expression der genannten Enzymaktivitäten in einer Wirtszelle zu vermitteln.10. A vector containing the expression cassette according to claim 9, which is suitable for mediating the expression of said enzyme activities in a host cell.
11. Verfahren zur biotechnologischen Herstellung von C2 - C6- Körpern als Produkt aus Kohlenstoffdioxid als Substrat und unter11. A method for the biotechnological production of C2 - C6 bodies as a product of carbon dioxide as substrate and under
Assimilierung von Wasserstoff, enthaltend die Schritte:Assimilation of hydrogen, containing the steps:
a) Bereitstellen einer in einem der Ansprüche 1 bis 8 definierten transgenen biologischen Zelle;a) providing a transgenic biological cell as defined in any one of claims 1 to 8;
b) Inkontaktbringen der Zelle mit dem Substrat;b) contacting the cell with the substrate;
c) Kultivieren der Zelle unter Bedingungen, unter denen Substrat umgesetzt und das Produkt gebildet wird;c) culturing the cell under conditions in which substrate is reacted and the product is formed;
d) Isolieren des Produkts.d) isolating the product.
12. Verwendung der in einem der Ansprüche 1 bis 8 definierten transgenen biologischen Zelle zur biotechnologischen Herstellung von Lactat oder Milchsäure aus Kohlenstoffdioxid und Wasserstoff.12. Use of the defined in any one of claims 1 to 8 transgenic biological cell for the biotechnological production of lactate or lactic acid from carbon dioxide and hydrogen.
13. Verwendung der in einem der Ansprüche 1 bis 8 definierten transgenen biologischen Zelle zur biotechnologischen Herstellung von Alkohol aus Kohlenstoffdioxid und Wasserstoff. 13. Use of the defined in any one of claims 1 to 8 transgenic biological cell for the biotechnological production of alcohol from carbon dioxide and hydrogen.
PCT/EP2008/008351 2007-10-02 2008-10-02 Biotechnological fixing of carbon dioxide WO2009046929A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007047206.6 2007-10-02
DE102007047206.6A DE102007047206B4 (en) 2007-10-02 2007-10-02 Biotechnological fixation of carbon dioxide

Publications (3)

Publication Number Publication Date
WO2009046929A2 true WO2009046929A2 (en) 2009-04-16
WO2009046929A3 WO2009046929A3 (en) 2009-05-22
WO2009046929A8 WO2009046929A8 (en) 2009-07-02

Family

ID=40149784

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/008351 WO2009046929A2 (en) 2007-10-02 2008-10-02 Biotechnological fixing of carbon dioxide

Country Status (2)

Country Link
DE (1) DE102007047206B4 (en)
WO (1) WO2009046929A2 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013018734A1 (en) 2011-07-29 2013-02-07 三井化学株式会社 Microorganism having carbon dioxide fixation pathway introduced thereinto
WO2014115815A1 (en) 2013-01-24 2014-07-31 三井化学株式会社 Microorganism having carbon dioxide fixation cycle introduced thereinto
WO2015005406A1 (en) 2013-07-09 2015-01-15 味の素株式会社 Method for manufacturing useful substance
CN105518148A (en) * 2013-06-29 2016-04-20 加利福尼亚大学董事会 Recombinant plants and microorganisms having a reverse glyoxylate shunt
RU2639507C2 (en) * 2012-04-24 2017-12-21 СиДжей ЧеилДжеданг Корпорейшн D-milk acid producing strain and its application
TWI652342B (en) 2012-12-21 2019-03-01 國立中興大學 Microrganism for forming fermentation products through fermentation of sugars
US10294505B2 (en) 2013-01-24 2019-05-21 Mitsui Chemcials, Inc. Microorganism for production of chemicals derived from acetyl-CoA
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
WO2020225346A1 (en) * 2019-05-09 2020-11-12 Enobraq Genetically modified bacterium for producing lactate from co2

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1088886A1 (en) * 1999-09-30 2001-04-04 Roche Diagnostics GmbH Methods for production of recombinant Holo-Cytrate Lyase
EP1672077A1 (en) * 2003-08-28 2006-06-21 Mitsubishi Chemical Corporation Process for producing succinic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1088886A1 (en) * 1999-09-30 2001-04-04 Roche Diagnostics GmbH Methods for production of recombinant Holo-Cytrate Lyase
EP1672077A1 (en) * 2003-08-28 2006-06-21 Mitsubishi Chemical Corporation Process for producing succinic acid

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DU CUIHONG ET AL: "Construction of a genetically engineered microorganism for CO2 fixation using a Rhodopseudomonas/Escherichia coli shuttle vector." FEMS MICROBIOLOGY LETTERS, Bd. 225, Nr. 1, 8. August 2003 (2003-08-08), Seiten 69-73, XP002510289 ISSN: 0378-1097 *
LIDSTROM ET AL: "Genetics of carbon metabolism in methylotrophic bacteria" FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, Bd. 87, Nr. 3-4, 1. Dezember 1990 (1990-12-01), Seiten 431-436, XP023922128 ISSN: 0378-1097 [gefunden am 1990-12-01] *
RANKIN C A ET AL: "Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum." PROTEIN SCIENCE : A PUBLICATION OF THE PROTEIN SOCIETY FEB 1993, Bd. 2, Nr. 2, Februar 1993 (1993-02), Seiten 197-205, XP002510386 ISSN: 0961-8368 *
TABITA F R: "Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms." MICROBIOLOGICAL REVIEWS JUN 1988, Bd. 52, Nr. 2, Juni 1988 (1988-06), Seiten 155-189, XP002510290 ISSN: 0146-0749 *
WENDISCH ET AL: "Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids" CURRENT OPINION IN MICROBIOLOGY, CURRENT BIOLOGY LTD, GB, Bd. 9, Nr. 3, 1. Juni 2006 (2006-06-01), Seiten 268-274, XP005484635 ISSN: 1369-5274 *
YUN NA-RAE ET AL: "A novel five-subunit-type 2-oxoglutalate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 22 MAR 2002, Bd. 292, Nr. 1, 22. März 2002 (2002-03-22), Seiten 280-286, XP002510018 ISSN: 0006-291X *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
TWI573869B (en) * 2011-07-29 2017-03-11 三井化學股份有限公司 Introducing microorganisms with a fixed cycle of carbon dioxide
WO2013018734A1 (en) 2011-07-29 2013-02-07 三井化学株式会社 Microorganism having carbon dioxide fixation pathway introduced thereinto
US9822387B2 (en) 2011-07-29 2017-11-21 Mitsui Chemicals, Inc. Microorganism having carbon dioxide fixation pathway introduced thereinto
RU2639507C2 (en) * 2012-04-24 2017-12-21 СиДжей ЧеилДжеданг Корпорейшн D-milk acid producing strain and its application
TWI652342B (en) 2012-12-21 2019-03-01 國立中興大學 Microrganism for forming fermentation products through fermentation of sugars
US10294505B2 (en) 2013-01-24 2019-05-21 Mitsui Chemcials, Inc. Microorganism for production of chemicals derived from acetyl-CoA
KR101714943B1 (en) 2013-01-24 2017-03-09 미쓰이 가가쿠 가부시키가이샤 Microorganism having carbon dioxide fixation cycle introduced thereinto
US9828618B2 (en) 2013-01-24 2017-11-28 Mitsui Chemicals, Inc. Microorganism having carbon dioxide fixation cycle introduced thereinto
KR20150041802A (en) * 2013-01-24 2015-04-17 미쓰이 가가쿠 가부시키가이샤 Microorganism having carbon dioxide fixation cycle introduced thereinto
WO2014115815A1 (en) 2013-01-24 2014-07-31 三井化学株式会社 Microorganism having carbon dioxide fixation cycle introduced thereinto
EP3013971A4 (en) * 2013-06-29 2016-11-30 Univ California RECOMBINANT PLANTS AND RECOMBINANT MICROORGANISMS WITH INVERTED GLYOXYLATE
CN105518148A (en) * 2013-06-29 2016-04-20 加利福尼亚大学董事会 Recombinant plants and microorganisms having a reverse glyoxylate shunt
WO2015005406A1 (en) 2013-07-09 2015-01-15 味の素株式会社 Method for manufacturing useful substance
EP3521433A1 (en) 2013-07-09 2019-08-07 Ajinomoto Co., Inc. Process for producing l-glutamic acid
WO2020225346A1 (en) * 2019-05-09 2020-11-12 Enobraq Genetically modified bacterium for producing lactate from co2
FR3095817A1 (en) * 2019-05-09 2020-11-13 Enobraq Genetically modified bacteria for improved production of lactate from CO2

Also Published As

Publication number Publication date
WO2009046929A8 (en) 2009-07-02
DE102007047206A1 (en) 2009-04-09
DE102007047206B4 (en) 2016-08-11
WO2009046929A3 (en) 2009-05-22

Similar Documents

Publication Publication Date Title
JP6898365B2 (en) Recombinant microorganisms and how to use them
DE102007047206B4 (en) Biotechnological fixation of carbon dioxide
Hon et al. The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum
JP6445970B2 (en) Recombinant microorganisms and uses thereof
US8945888B2 (en) Method for producing high amount of glycolic acid by fermentation
De Tissera et al. Syngas biorefinery and syngas utilization
CN1097632C (en) A mutant E. coli strain with increased succinic acid production
WO2003100013A2 (en) NON-REVERTIBLE β-OXIDATION BLOCKED CANDIDA TROPICALIS
CN112204146B (en) Acid-tolerant yeast having an inhibited ethanol production pathway and method for producing lactic acid using same
JP2012506716A (en) Microaerobic culture for converting glycerol to chemicals
JP2022008224A (en) Recombinant acid-resistant yeast with increased lactic acid production capacity
RU2422526C2 (en) METHOD OF PRODUCING SUCCINIC ACID WITH USING Yarrowia YEAST
US20170159083A1 (en) Arginine supplementation to improve efficiency in gas fermenting acetogens
JP4473219B2 (en) Biocatalyst for D-lactic acid production
KR20150121789A (en) Recombinant microorganism having enhanced butanediol producing ability and method for producing butanediol using the same
DE102007059248A1 (en) Recombinant cell capable of converting bicarbonate to organic compounds utilizes a 16-step metabolic pathway starting with acetyl coenzyme A
DE102007048625B4 (en) Biotechnological production of crotonic acid
WO2022156857A1 (en) Process for preparing 2,4-dihydroxybutyrate or l-threonine using a microbial metabolic pathway
DE102021113602A1 (en) GMO YEAST FOR THE BIOTECHNOLOGICAL PRODUCTION OF Succinic ACID FROM GLYCEROL
DE10312775B4 (en) Method and microorganism for the microbial production of L-alanine
US20250084445A1 (en) Organisms and methods of use thereof
US20250043254A1 (en) Particularly suitable 3-desoxyarabinoheptulosanate-7-phosphate synthase for the fermentative preparation of ortho-aminobenzoic acid
JP2006513693A (en) Non-revertible β-oxidation-blocking Candida tropicalis
WO2016164339A2 (en) Compositions and methods for the conversion of short-chained carboxylic acids to alcohols using clostridial enzymes
DE102007045093A1 (en) Process for the preparation of 2-methyl-1,2-dihydroxypropane

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08802753

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 08802753

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载