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WO2018127544A1 - Utilisation d'enzymes radicalaires s-adénosyl méthionines (sam) pour l'introduction d'acides α-céto-β3-aminés dans des (poly)peptides - Google Patents

Utilisation d'enzymes radicalaires s-adénosyl méthionines (sam) pour l'introduction d'acides α-céto-β3-aminés dans des (poly)peptides Download PDF

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WO2018127544A1
WO2018127544A1 PCT/EP2018/050225 EP2018050225W WO2018127544A1 WO 2018127544 A1 WO2018127544 A1 WO 2018127544A1 EP 2018050225 W EP2018050225 W EP 2018050225W WO 2018127544 A1 WO2018127544 A1 WO 2018127544A1
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amino acid
seq
group
rsam
acid sequence
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Jörn PIEL
Brandon MORINAKA
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Eth Zurich
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01006—Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase

Definitions

  • the present invention relates to the use of a radical S-adenosyl methionine (rSAM) enzyme in a method for introducing at least one a-keto-3 ⁇ 4 3 -amino acid into (poly)peptide substrates comprising one or more of amino acid motif XYG, wherein X is any natural or non-natural amino acid, Y is tyrosine and G is glycine. Furthermore, the present invention relates to a method for introducing at least one a-keto-3 ⁇ 4 3 -amino acid into (poly)-peptides comprising said XYG motif using a radical S-adenosyl methionine (rSAM) enzyme.
  • rSAM radical S-adenosyl methionine
  • ⁇ -amino acids are widely distributed in nature and are present in natural product-derived drugs such as penicillin, taxol and cocaine.
  • the installation of the ⁇ -amino acids in peptides offers, e.g., greater stability (for example from protease-mediated degradation) and can confer structural features distinct from all L-amino acid-containing peptides or natural products, resulting in unique functions and bioactivities.
  • Known methods to incorporate ⁇ -amino acids into peptides generally rely on total synthesis, for example, by condensation of monomers in solid- phase synthesis.
  • a second approach relies on in vitro translation, which usually suffers from low incorporation efficiency.
  • ribosomally synthesized peptides and proteins are comprised of a-amino acids.
  • No post-translational modifying enzymes are known that incorporate ⁇ -amino acids into ribosomal products.
  • Biosynthetic routes to ⁇ -amino acid-containing peptides typically utilize non-ribosomal peptide synthetases (NRPS) that act on free amino acids or peptide residues.
  • NRPS non-ribosomal peptide synthetases
  • These enzymes are very large multimodular enzymes and are difficult to manipulate for bioengineering or biotechnological applications.
  • the loading of amino acids onto NRPS usually occurs specifically for certain amino acids.
  • the NRPS-type machinery is limited to small peptides with typically less than 15 residues. Therefore, incorporation of ⁇ - amino acids into proteins by NRPSs is not possible.
  • the above objective is solved by the use of a radical S-adenosyl methionine (rSAM) enzyme in a method for introducing at least one a-keto-IS 3 -amino acid into (poly)- peptide substrates comprising one or more of amino acid motif XYG, wherein X is any natural or non-natural amino acid, Y is tyrosine and G is glycine.
  • rSAM radical S-adenosyl methionine
  • radical S-adenosyl methionine (rSAM) enzymes have the capacity to incorporate different types of a-keto-IS 3 -amino acids into diverse (poly)peptide substrates that comprise one or more of amino acid motif XYG.
  • rSAM enzymes are monomeric enzymes of up to 50 kDa and can be overexpressed in high yields in many cells, e.g. in E. coli (see below).
  • peptides generated by NRPSs require huge enzyme complexes and are typically quite specific to result in either a single product or a few closely related analogs only.
  • Preferred rSAM enzymes for use in the present invention were identified by their surrogate substrate nifll precursor peptides within ribosomally synthesized and post-translationally modified peptide (RiPP) natural product gene clusters.
  • Gene clusters containing NHLP- or Nifll- type precursors are considered a new natural product family (see Freeman et al. Science 338, 387-90 (2012) and Haft et al. BMC Biology 8, 70 (2010)) termed "proteusins".
  • proteusin biosynthetic gene clusters are widespread in bacteria, their biosynthetic end products and functions are currently unknown with the exception of the cytotoxic pore-forming polytheonamides (see Hamada et al. Tetrahedron Lett.
  • a-keto ⁇ 3 -amino acid-comprising (poly)peptide products can conveniently be generated according to the present invention by in vivo co-expression of a (poly)peptide substrate with the rSAM enzymes in bacteria and also other organisms, e.g. yeast, plant cells, mammalian cells or insect cells.
  • the rSAM enzymes described herein also introduce a keto functionality that is not present in proteinogenic amino acids.
  • the introduction of a keto functionality into ribosomally synthesized (poly)peptides is of interest, e.g.
  • non-selective chemical oxidation such as sodium periodate to oxidize all side chains of serine
  • introduction of non-canonical amino acids by an unnatural amino acid mutagenesis strategy based on recoding of stop codons.
  • the pro- teusin cluster e.g. from Pleurocapsa sp. PCC 7319, containing genes for two substrates (e.g. plpA2 (SEQ ID NOs: 114 and 115) and plpA3 (SEQ ID NOs: 116 and 117), an rSAM SPASM protein (e.g. plpX (SEQ ID NOs: 118 and 119), and optionally an associated protein (e.g. plpY (SEQ ID NOs: 120 and 121) can be cloned into expression vectors for expression in E. coli.
  • the associated protein e.g.
  • plpY is only optional, e.g. to increase the enzymatic efficiency of the rSAM and is not required for practicing the present invention.
  • the substrate peptides e.g. plpA2 and plpA3
  • the rSAM enzyme and the optional associated protein e.g. pip X and plpY
  • the substrate peptide genes can be individually transformed with the rSAM enzyme and the optional associated protein (e.g. plpX and plpY), e.g. into E.
  • coli and protein overexpression can be carried out in standard medium.
  • purification e.g. Ni-affinity purification of the N-terminally hexahistidine-tagged (NHis6) peptides, and in vitro cleavage of leader, e.g. with a protease
  • the core (poly)peptide comprising the a-keto ⁇ 3 -amino acid(s) can be analyzed, e.g. by MALDI-MS, and optionally the a-keto ⁇ -amino acid(s) can be reduced to the corresponding ⁇ -amino acid(s) using standard chemical methods, e.g. using sodium borohydride.
  • the rSAM enzyme for use in the present invention excises a structure from the XYG motif that has a mass of C 8 H 9 NO, as determined by high resolution mass spectrometry and is attributed to the tyrosine residue.
  • the structures of the transformed substrates are products containing the a-keto ⁇ 3 -amino acid.
  • the XYG motif is conserved among a wide range of precursors, e.g.
  • precursors carrying more than one XYG motif can be converted to products with several a-keto ⁇ 3 -amino acids after expression of the rSAM enzyme in E. coli.
  • ⁇ -amino acid refers to any non-natural or non-conventional amino acid, preferably to any proteinogenic amino acid, more preferably to any of the 20 standard amino acids, that has its amino group bonded to the ⁇ -carbon rather than the a carbon.
  • a-keto ⁇ 3 -amino acid refers to any non-natural or non-conventional amino acid, preferably to any proteinogenic amino acid, more preferably to any of the 20 standard amino acids, that has a keto group at the a carbon and its amino group bonded to the 3- ⁇ 3 ⁇ rather than the a carbon.
  • polypeptide as used herein, is meant to encompass peptides, polypeptides, oligopeptides and proteins that comprise two or more amino acids linked covalently through peptide bonds. The term does not refer to a specific length of the product.
  • the term (poly)- peptide includes (poly)peptides with post-translational modifications, for example, glycosylates, acetylations, phosphorylations and the like, as well as (poly)peptides comprising non- natural or non-conventional amino acids and functional derivatives as described below.
  • non-natural or non-conventional amino acid refers to naturally occurring or naturally not occurring unnatural amino acids or chemical amino acid analogues, e.g. D-amino acids, ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehyd roamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or para-aminobenzoic acid.
  • D-amino acids e.g. D-amino acids, ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehyd roamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or para-aminobenzoic acid.
  • Non-conventional amino acids also include compounds which have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as ⁇ -alanine, y-amino butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD) , amino- methyl benzoic acid and others well known in the art.
  • BTD bicyclic dipeptide
  • Statine-like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art may also be used.
  • a non limiting list of non-conventional amino acids which may be comprised in the (poly)peptide and their standard abbreviations (in brackets) is as follows: a-aminobutyric acid (Abu), L-N-methylalanine (Nmala), ⁇ -amino-a-methylbutyrate (Mgabu), L-N-methylarginine (Nmarg), aminocyclopropane (Cpro), L-N-methylasparagine (Nmasn), carboxylate L-N-methyl- aspartic acid (Nmasp), aniinoisobutyric acid (Aib), L-N-methylcysteine (Nmcys), aminonorbornyl (Norb), L-N-methylglutamine (Nmgln), carboxylate L-N-methylglutamic acid (Nmglu), cyclohexyl- alanine (Chexa), L-N-methylhistidine (Nmhis), cyclopentylalanine
  • Nmaabu D-a-methylleucine (Dmleu), a-napthylalanine (Anap), D-a-methyllysine (Dmlys), N- benzylglycine (Nphe), D-a-methylmethionine (Dmmet), N-(2-carbamylethyl)glycine (Ngln), D-a- methylornithine (Dmorn), N-(carbamylmethyl)glycine (Nasn), D-a-methylphenylalanine (Dmphe), N-(2-carboxyethyl)glycine (Nglu), D-a-methylproline (Dmpro), N-(carboxymethyl)glycine (Nasp), D-a-methylserine (Dmser), N-cyclobutylglycine (Ncbut), D-a-methylthreonine (Dmthr), N-cyclo- heptylglycine (
  • the rSAM enzyme for use in the present invention is a peptide radical SAM maturase, preferably a nifll class peptide radical SAM maturase 3.
  • the nifll-class peptide radical SAM maturase 3 belongs to the conserved protein domain family rSAM_nifll_3 (nifll-class peptide radical SAM maturase 3; IPR026482) or the rad_SAM_trio family (radical SAM GDL-associated; IPR023820) as defined by the National Center for Biotechnology Information (NCBI).
  • rSAM_nifll_3 are radical SAM enzymes that often occur co-clustered together with nifll-related ribosomal natural product (RNP) precursors described by TIGRFAMs model TIGR03798.
  • RNP ribosomal natural product
  • rad_SAM_trio radical SAM enzymes that often occur co-clustered together with DUF1843-domain RNP precursors carrying a YXioGDL motif and described by Pfam model PF08898 (see Haft et al. Nucleic Acids Res. 31, 371-3 (2003); Haft and Basu, J. Bacteriol. 193, 2745-2755 (2011); NCBI database on
  • the rSAM enzyme for use in the present invention comprises (A) an amino acid sequence according to Formula (I) (SEQ ID NO: 1) or (II) (SEQ ID NO: 2)
  • ⁇ - ⁇ 20 and Zi-Z 20 each denote amino acids
  • Xi is selected from the group consisting of Y, H, F and W, preferably Y and H;
  • X 2 is selected from the group consisting of Y, R and H;
  • X 3 is selected from the group consisting of R, K and Q, preferably R;
  • X 4 is selected from the group consisting of I, T and V, preferably I and T;
  • X 5 is selected from the group consisting of R, S and K, preferably R and S;
  • X 6 is selected from the group consisting of H, Y, F and W, preferably H and Y;
  • X 7 is selected from the group consisting of A and S, preferably A;
  • X 8 is selected from the group consisting of V, I and L, preferably V;
  • X 9 is selected from the group consisting of W, Y and F, preferably W;
  • Xio is selected from the group consisting of E, Q, D and K, preferably E;
  • Xii is selected from the group consisting of I, L, V and M, preferably I and L;
  • Xi2 is selected from the group consisting of T and S, preferably T;
  • Xi3 is selected from the group consisting of L, M, I and V, preferably L;
  • Xi4 is selected from the group consisting of K, R, E and Q, preferably K;
  • Xi5 is C
  • Xi6 is selected from the group consisting of N and D, preferably N;
  • Xi7 is selected from the group consisting of L, M, I and V, preferably L;
  • Xi8 is selected from the group consisting of A and S, preferably A;
  • X 2 o is selected from the group consisting of S, Q, E and K, preferably S and Q;
  • Zi is selected from the group consisting of T, D, T, E and N, preferably T and D;
  • Z 2 is selected from the group consisting of R, P, N and A, preferably R, P and A;
  • Z 3 is selected from the group consisting of R, Q, K and L, preferably R;
  • Z 5 is selected from the group consisting of A and S, preferably A;
  • Z 6 is selected from the group consisting of R, K and Q, preferably R;
  • Z 7 is selected from the group consisting of Y, F, H and W, preferably Y;
  • Z 8 is selected from the group consisting of L, M, I and V, preferably L;
  • Z 9 is selected from the group consisting of F, H, S and Y, preferably H, F and S;
  • Z10 is selected from the group consisting of D, E and A, preferably D and E;
  • Zn is selected from the group consisting of D, S and T, preferably T;
  • Z12 is selected from the group consisting of D, E and N, preferably D;
  • Zi3 is selected from the group consisting of Y, F, L and M, preferably Y, F and L;
  • Zi4 is selected from the group consisting of K, Q, R and E, preferably Q. and K;
  • Zi5 is selected from the group consisting of R, K and Q, preferably R;
  • Zi6 is selected from the group consisting of Y, F and W, preferably Y and F;
  • Zi7 is selected from the group consisting of V, I and L, preferably V;
  • Zi9 is selected from the group consisting of V, I and L, preferably V;
  • Z 2 o is selected from the group consisting of H and Y, preferably H; or
  • (C) a functional fragment and/or functional derivative of (A) or (B), preferably a functional fragment of at least 10 amino acids, more preferably at least 15 amino acids of (A) or (B).
  • the percentage identity of related amino acid molecules can be determined with the assistance of known methods. In general, special computer programs are employed that use algorithms adapted to accommodate the specific needs of this task. Preferred methods for determining identity begin with the generation of the largest degree of identity among the sequences to be compared. Preferred computer programs for determining the identity among two amino acid sequences comprise, but are not limited to, TBLASTN, BLASTP, BLASTX, TBLASTX (Altschul et al., J. Mol.
  • the BLAST programs can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST handbook, Altschul et al., NCB NLM NIH Bethesda, MD 20894).
  • NCBI National Center for Biotechnology Information
  • the ClustalW program can be obtained from
  • the term "functional derivative" of a (poly)peptide of the present invention is meant to include any (poly)peptide or fragment thereof that has been chemically or genetically modified in its amino acid sequence, e.g. by addition, substitution and/or deletion of amino acid residue(s) and/or has been chemically modified in at least one of its atoms and/or functional chemical groups, e.g. by additions, deletions, rearrangement, oxidation, reduction, etc. as long as the derivative still has at least some rSAM activity to a measurable extent, e.g. of at least about 1 to 10%, preferably 10 to 50% rSAM activity of the original unmodified (poly)peptide of the invention.
  • a "functional fragment" of the invention is one that forms part of a
  • polypeptide or derivative of the invention still has at least some rSAM activity to a measurable extent, e.g. of at least about 1 to 10%, preferably 10 to 50% rSAM activity of the original unmodified (poly)peptide of the invention.
  • amino acid sequence of Formula (I) and (II) are based on the sequences disclosed by TIGFRAMs TIGR04103 (Formula (I) above) and TIGR03913 (Formula (II) above) with the specific preferred amino acids defined for ⁇ - ⁇ 20 and ⁇ - ⁇ 2 ⁇
  • the rSAM enzyme for use in the present invention comprises at least one motif selected from the group consisting of
  • motif CXXXCXXC (SEQ ID NO: 3), wherein X is any natural amino acid and wherein the motif CXXXCXXC (SEQ ID NO: 3) is preferably comprised in an N-terminal radical SAM domain;
  • motif CX 9 _i 5 GX 4 C (SEQ ID NO: 6) reads on a motif consisting of amino acid C, 9 to 15 natural amino acids, amino acid G, 4 natural amino acids and amino acid C.
  • the rSAM enzyme for use in the present invention comprises
  • rSAM enzyme further comprises
  • the rSAM enzyme for use in the present invention comprises
  • the rSAM enzyme for use in the present invention comprises an amino acid sequence selected from the group of
  • sequences listed in any of SEQ ID NOs: 12 to 54 preferably SEQ ID NOs: 39 and 40, or an amino acid sequence having an amino acid sequence identity of at least 70 or 80 %, preferably at least 90 or 95 % with the amino acid sequences in any of SEQ ID NOs: 12 to 54;
  • amino acid sequence having an amino acid sequence identity of at least 80 %, preferably at least 90 or 95 % with the amino acid sequences in any of SEQ ID NOs: 55 to 113.
  • the present invention is directed to a use of a recombinant vector comprising a nucleic acid encoding an rSAM enzyme as defined above in a method for introducing at least one a-keto-IS 3 -amino acid into (poly)peptides comprising one or more amino acid motifs XYG, wherein X is any natural or non-natural amino acid, Y is tyrosine and G is glycine, preferably a viral or episomal vector, more preferably a vector selected from the group consisting of lentivirus vector, adenovirus vector, baculovirus vector, bacterial vector and yeast vector.
  • the viral vector is a lentivirus vector (see for example System Biosciences, Mountain View, CA, USA), adenovirus vector (see for example ViraPower Adenoviral Expression System, Life Technologies, Carlsbad, CA, USA), baculovirus vector (see for example Bac-to-Bac Expression Kit Handbook, Invitrogen Corporation, Carlsbad, Calif.), bacterial vector (see for example Novagen, Darmstadt, Germany)) or yeast vector (see for example ATCC Manassas, Virginia).
  • Vector construction including the operable linkage of a coding sequence with a promoter and other expression control sequences, is within the ordinary skill in the art.
  • a host cell expressing an rSAM enzyme as defined above preferably comprising a recombinant vector as defined above, in a method for introducing at least one a-keto-IS 3 -amino acid into (poly)peptides comprising one or more amino acid motifs XYG, wherein X is any natural or non-natural amino acid, Y is tyrosine and G is glycine, preferably a host cell selected from the group consisting of yeast cells, preferably Saccharomyces cerevisiae cells (see for example Methods in Enzmology, 350, 248, 2002), and Pichia pastoris cells (see for example Pichia Expression Kit Instruction Manual, Invitrogen Corporation, Carlsbad, Calif.); bacterial cells, preferably E.
  • coll cells preferably BL21(DE3), K-12 and derivatives (see for example Applied Microbiology and Biotechnology, 72, 211, 2006), and Bacillus subtilis cells, preferably 1012 wild type, 168 Marburg or WB800N (see for example Westers et al., (2004) Mol. Cell. Res. Volume 1694, Issues 1-3 P:299-310); plant cells, preferably Nicotiana tabacum, and Physcomitrella patens (see e.g. Lau and Sun, Biotechnol Adv.
  • NIH-3T3 mammalian cells see for example Sambrook and Russell, 2001
  • insect cells preferably sf9 insect cells (see for example Bac-to-Bac Expression Kit Handbook, Invitrogen Corporation, Carlsbad, Calif.).
  • rSAM-associated protein plpY (SEQ ID NO: 121) is optional but can significantly contribute to the efficiency of rSAM enzyme activity in a method for introducing at least one a-keto-IS 3 -amino acid into (poly)peptide substrates comprising one or more of amino acid motif XYG.
  • the present invention is also directed to an rSAM-associated protein comprising an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 121, (b) an amino acid sequence having an amino acid sequence identity of at least 70 or 80 %, preferably at least 90 or 95 % with SEQ ID NO: 121, and (c) a functional fragment and/or functional derivative of (a) or (b), preferably a functional fragment of at least 30 amino acids, more preferably at least 45 amino acids of (a) or (b).
  • the rSAM-associated protein is preferably for use in combination with an rSAM enzyme as described above in a method for introducing at least one a-keto-IS 3 -amino acid into (poly)peptide substrates comprising one or more of amino acid motif XYG. More preferably, the rSAM-associa- ted protein is expressed by a recombinant vector and/or host cell also expressing the rSAM enzyme as described above.
  • the present invention relates to a method for introducing at least one a- keto-IS 3 -amino acid into (poly)-peptides comprising the steps of:
  • polypeptide substrate in step (iii), preferably both, optionally together with the rSAM-associated protein of optional step (ii), of the above method are provided in the form of a host cell, more preferably are all co-expressed in the host cell as defined above.
  • the host cell for use in step (i) of the above method is an E. coli host cell.
  • step (iv) is followed by step (v), wherein the keto-functionality resulting from step (iv) is reduced chemically, preferably by sodium
  • borohydride or is converted to an imine, preferably the methoxyamine.
  • E. coli expressing an rSAM enzyme as defined above is cultured in a rich medium such as TB medium, LB or YT medium.
  • a culture in the above medium preferably in an Erienmeyer flask or ultra-yield flask, is inoculated with an overnight culture at a concentration of preferably about 1:100.
  • the culture is grown at about 37°C and shaken at, e.g. about 250 RPM until, e.g. an OD 600 of about 1.2-2.0.
  • the culture is cooled, e.g. on ice, and induced with, e.g. IPTG (preferably about ImM final concentration).
  • the culture is shaken at, e.g.
  • the cells are collected by centrifugation, lysed, and the substrate subjected to purification, preferably Ni-affinity purification.
  • purification preferably Ni-affinity purification.
  • the product(s) are verified, e.g. by mass spectrometry of the full length or digested precursors, e.g. NHis-precursors.
  • the keto-functionality may be reduced (e.g. by sodium borohydride) or converted to the imine, preferably the methoxyamine.
  • the present invention is directed to isolated and purified nucleic acids encoding the (poly)peptides for use in the present invention.
  • Fig. 1 a) pip gene cluster encoding precursors (plpAl, A2, and A3), rSAM epimerase (plpD), rSAM excision enzyme (plpX) and associated protein (plpY). b) Protein sequences for core peptides of precursors PlpA2 (SEQ ID NO: 122), PlpA3 (SEQ ID NO: 123), PlpA3-9 (SEQ ID NO: 124) and PcpA (SEQ ID NO: 125).
  • Fig. 3 MS 2 spectra results for 1 (SEQ ID NO: 122). ⁇ Ex' indicates no excision. '-1 Tya' indicates loss of 'tyramine' (C 8 H 9 NO) from the corresponding fragment.
  • Fig. 4 MS 2 spectra for 2 (SEQ ID NO: 122). ⁇ Ex' indicates no excision. '-1 Tya' indicates loss of 'tyramine' (C 8 H 9 NO) from the corresponding fragment.
  • Fig. 5 MS 2 spectra for 3 (SEQ ID NO: 126). ⁇ Ex' indicates no excision. '-1 Tya' indicates loss of 'tyramine' (C 8 H 9 NO) from the corresponding fragment.
  • Fig. 6 MS 2 spectra for 4 (SEQ ID NO: 126). ⁇ Ex' indicates no excision. '-1 Tya' indicates loss of 'tyramine' (C 8 H 9 NO) from the corresponding fragment.
  • Fig. 7 Sodium borohydride reduction of a mixture of 1 (SEQ ID NO: 122) and 2 (SEQ ID NO:
  • Fig. 8 HMBC spectra for product 5 (SEQ ID NO: 127) showing key HMBC correlations to th keto and amide carbonyls of the a-keto ⁇ -amino acid.
  • Fig. 9 HMBC spectra for product 6 (SEQ ID NO: 127) showing key HMBC correlations to th keto and amide carbonyls of the a-keto ⁇ -amino acid.
  • Fig. 10 13 C spectra for product 6 (SEQ ID NO: 127) from feeding experiments with methionine.
  • Fig. 11 13 C spectra for product 6 (SEQ ID NO: 127) from feeding experiments with methionine.
  • Fig. 12 Reaction catalyzed by PIpX.
  • Fig. 13 Results for all PlpA3-Fx mutants. Shown is the peptide fragment (SEQ ID NOs: 128 to 143) affected by mutation and the respective detection of conversion.
  • th( gene was co-expressed with either of the two precursor genes plpA2 (SEQ ID NOs: 114 and 115) and plpA3 (SEQ ID NOs: 116 and 117) located upstream.
  • the translated precursors contain, in addition to an N-terminal leader region of the Nifll family, predicted core regions of 25 and 23 aa, respectively (Fig. lb).
  • the Nifll precursor genes (plpAl and plpA2) were individually cloned with N-terminal His6-tags and a Factor Xa site at the interface of the leader and core and inserted into pACYCDuet-1.
  • the rSAM gene (plpX) was cloned into MCSII of pRSFDuet-1 and constructs were transformed into E. coli BL21 DE3 for protein expression (Fig 2). Under these conditions, transformation of the precursors was not observed. More detailed analysis of the pip cluster (Fig. la) revealed a small conserved gene, plpY (SEQ ID NOs: 120 and 121), located downstream of plpX, an architecture also preserved in other clusters with plpX homologs.
  • HMBC Hetero- nuclear Multiple Bond Correlation
  • PlpA3-Fx was used to investigate the origin of the ⁇ -amino acid moiety by feeding of various 13 C-labeled amino acids to E. coli expression cultures.
  • labels of [l- 13 C] Met, [U- 13 C]Met, [l- 13 C]Tyr, and [U- 13 C]Tyr were detected by MS in the peptide products.
  • NMR-based characterization of the purified core fragment 6 revealed enhancements of carbon signals that were consistent with Met remaining fully intact (Fig. 10), while only CI of Tyr is retained and accounts for the amide carbonyl in the product (Fig. 11).
  • PCC 7327 was co- expressed with its cognate excisase gene partners pcpX and pcpY. The excision reaction was detected at two of the three YG motifs. With a translated core containing 64 aa and three predicted YG motifs, the pep pathway generates a giant natural product that may exceed the size of all specialized metabolites reported to date.
  • Factor Xa protease was purchased from Merck (USA). Restriction enzymes and GluC were purchased from New England Biolabs (USA). Thermo Scientific (USA) Phusion ® DNA polymerase and T4 DNA ligase were used for all PCRs and ligations, respectively. DNA primers were obtained from Microsynth (Switzerland) or Thermo Scientific (USA). Antibiotics (chloramphenicol for pACYCDuet-1 and kanamycin for pRSFDuet-1) were used at a concentration of 25mg/mL in solid and liquid medium.
  • Expression vectors containing NHis 6 -precursor genes were constructed as follows. Mini-preps derived from previously reported plasmids (plpAl-Fx, plpA2-Fx, and p/p3-Fx in pET-28b) (see Morinaka, B. I. et al. Angew. Chem. Int. Ed. 53, 8503-8507 (2014)) containing NHis 6 -precursor genes containing a Factor Xa site (IDGR) at the interface of leader and core peptide were digested with Ncol and EcoRI.
  • IDGR Factor Xa site
  • the precursor peptide inserts were gel-purified, ligated into MCSI of pACYCDuet-1 to give pAlFxACYC, pA2FxACYC, and pA3FxACYC. These precursor constructs were sequence verified. Constructs for the excision enzyme (PlpX) and associated protein (PlpY) were constructed as follows. The gene for the excision enzyme was amplified by PCR (primers PlpX_F,
  • ATCTCTCG AGTTACTTTG CT A AAG CGTA AG C AG A (SEQ ID NO: 145)) and products were gel-purified, digested with Ndel and Xhol and ligated into MCSII of pRSFDuet-1 to give plasmid pXRSF.
  • the gene for the associated protein was amplified by PCR (PlpY_F, GCGAACTCATGA ACTCTAATCAAATACCAAATAAA (SEQ ID NO: 146) and PlpY_R, GCGCAGCTGT- TATGTCAGAAAATTGCT (SEQ ID NO: 147)), gel-purified, digested with BspHI and Sail, ligated into MCSI of pXRSF (cut with Ncol and Sail) to give pXYRSF, and the insert confirmed by sequencing.
  • Precursor constructs were transformed and expressed in E. coli BL21(DE3) cells alone and with pXRSF or pXYRSF. Proteins containing a Factor Xa cleavage site are denoted with an 'Fx'.
  • TB medium (30 mL) containing appropriate antibiotics was inoculated with 300 ⁇ _ overnight culture grown in LB. The cells were grown at 37°C at 250 rpm until an OD 600 of ⁇ 1.6-2.0, cooled on ice for 30 min, induced with IPTG (1 mM final concentration), then shaken for 24 hours (250 rpm, 16 °C). The cells were collected by centrifugation (3,220 x g, 10 min). Proteins were purified using Ni-NTA resin (Macherey-Nagel (Germany)) according to the manufacturer's protocol. 10% glycerol was added to the lysis, wash and elution buffers.
  • Proteins were adsorbed using 0.5 mL Ni-NTA resin, and eluted with 2.5 mL (250 mM imidazole, 50 mM sodium phosphate, 300 mM NaCI, and 10% (v/v) glycerol, pH 8). Elution fractions were desalted on a PD-10 column, digested with Factor Xa or trypsin, and ana- lyzed by LC-MS and MALDI.
  • LC-MS conditions column: Kinetex C18-XB, 2.6 ⁇ , 150 x 4.6 mm; flow rate: 1.0 mL/min; mobile phase/gradient: 95:5 A/B for 5 minutes ramped to 40:60 A/B over 30 minutes.
  • SEQ ID NO: 12 (70% TIGR03913, >WP_052261552)
  • SEQ ID NO: 15 (70% TIGR03913, >OCW56221)
  • SEQ ID NO: 19 (70% TIGR03913, >WP_050043969)
  • SEQ ID NO: 20 (70% TIGR03913, >WP_020539729)
  • SEQ ID NO: 24 (70% TIGR03913, >WP_062765523)
  • SEQ ID NO: 26 (70% TIGR04103, >WP_020737613)
  • SEQ ID NO: 28 (70% TIGR04103, >KIG18351)
  • SEQ ID NO: 29 (70% TIGR04103, >WP_006974883)
  • SEQ ID NO: 30 (70% TIGR04103, >WP_012234464)
  • SEQ ID NO: 32 (70% TIGR04103, >WP_010607032)
  • SEQ ID NO: 33 (70% TIGR04103, >WP_010607027)
  • SEQ ID NO: 34 (70% TIGR04103, >WP_054014533)
  • SEQ ID NO: 38 (70% TIGR04103, >SEA53645)
  • SEQ ID NO: 45 (70% TIGR04103, >WP_015929579)
  • SEQ ID NO: 46 (70% TIGR04103, >SFE54945)
  • SEQ ID NO: 48 (70% TIGR04103, >SFE67100)
  • SEQ ID NO: 50 (70% TIGR04103, >WP_006972642)
  • SEQ ID NO: 52 (70% TIGR04103, >WP_006969608)
  • SEQ ID NO: 54 (70% TIGR04103, >AKV02060)
  • SEQ ID NO: 56 (80% TIGR03913, >WP_052261552)
  • SEQ ID NO: 70 (80% TIGR03913, >WP_006753568)
  • SEQ ID NO: 72 (80% TIGR03913, >WP_062765523)
  • SEQ ID NO: 82 (80% TIGR04103, >WP_012234464)
  • SEQ ID NO: 83 (80% TIGR04103, >WP_002625456)
  • SEQ ID NO: 90 (80% TIGR04103, >WP_002708735) MSTQLRQRRTYAVWEITLKCNLACQHCGSRAGEARQDELSTAEALDLVQQMAEAGIGEVTLIGGEAFLRKD

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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne l'utilisation d'une enzyme radicalaire S-adénosyl méthionine (rSAM) dans un procédé d'introduction d'au moins un acide α-céto-β3-aminé dans des substrats (poly)peptidiques comprenant un ou plusieurs motifs acide aminé (XYG), X étant n'importe quel acide aminé d'origine naturelle ou synthétique, Y étant la tyrosine et G la glycine. En outre, la présente invention concerne un procédé d'introduction d'au moins un acide α-céto-β3-aminé dans des (poly)-peptides comprenant ledit motif (XYG) en utilisant une enzyme radicalaire S-adénosyl méthionine (rSAM).
PCT/EP2018/050225 2017-01-06 2018-01-05 Utilisation d'enzymes radicalaires s-adénosyl méthionines (sam) pour l'introduction d'acides α-céto-β3-aminés dans des (poly)peptides WO2018127544A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4159743A1 (fr) * 2021-09-30 2023-04-05 ETH Zurich Procédés de préparation de composés de pyridazine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030113882A1 (en) 1998-11-24 2003-06-19 Wisconsin Alumni Research Foundation Methods for the preparation of beta-amino acids
WO2007047680A2 (fr) * 2005-10-14 2007-04-26 Cargill, Incorporated Augmentation de l'activite des enzymes a radical s-adenosyle methionine (sam)

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US20030113882A1 (en) 1998-11-24 2003-06-19 Wisconsin Alumni Research Foundation Methods for the preparation of beta-amino acids
WO2007047680A2 (fr) * 2005-10-14 2007-04-26 Cargill, Incorporated Augmentation de l'activite des enzymes a radical s-adenosyle methionine (sam)

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Title
"NCBI", Database accession no. rad_SAM_trio
"NCBI", Database accession no. rSAM_nif11_3
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NCB NLM NIH BETHESDA
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 72, 2006, pages 211
CLARISSA MELO CZEKSTER ET AL: "In Vivo Biosynthesis of a β-Amino Acid-Containing Protein", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 138, no. 16, 27 April 2016 (2016-04-27), US, pages 5194 - 5197, XP055369605, ISSN: 0002-7863, DOI: 10.1021/jacs.6b01023 *
CZEKSTER ET AL., JACS, vol. 138, 2016, pages 5194 - 5197
DATABASE EMBL [online] 23 November 2015 (2015-11-23), "Labilithrix luteola Radical SAM, Pyruvate-formate lyase-activating enzyme like protein", XP002778916, Database accession no. AKU99181 *
DATABASE GenPept [online] 27 October 2016 (2016-10-27), "radical SAM/SPASM domain-containing protein [Ruegeria sp. ANG-S4]", XP002778915, retrieved from NCBI Database accession no. WP_052261552 *
FREEMAN ET AL., SCIENCE, vol. 338, 2012, pages 387 - 390
FUKUHARA, K. ET AL., ORG. LETT., vol. 17, 2015, pages 2646 - 2648
HAFT ET AL., BMC BIOLOGY, vol. 8, 2010, pages 70
HAFT ET AL., J. BACTERIOL., vol. 193, 2011, pages 2745 - 2755
HAFT ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages 371 - 373
HAFT; BASU, J. BACTERIOL., vol. 193, 2011, pages 2745 - 2755
HAMADA ET AL., TETRAHEDRON LETT., vol. 35, 1994, pages 719 - 720
LARKIN MA ET AL., BIOINFORMATICS, vol. 23, 2007, pages 2947 - 2948
LAU; SUN, BIOTECHNOL ADV., vol. 27, no. 6, 2009, pages 1015 - 1022
METHODS IN ENZMOLOGY, vol. 350, 2002, pages 248
MORINAKA, B. I. ET AL., ANGEW. CHEM. INT. ED., vol. 53, 2014, pages 8503 - 8507
WESTERS ET AL., MOL. CELL. RES., vol. 1694, no. 1-3, 2004, pages 299 - 310

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4159743A1 (fr) * 2021-09-30 2023-04-05 ETH Zurich Procédés de préparation de composés de pyridazine
WO2023052526A1 (fr) * 2021-09-30 2023-04-06 Eth Zurich Procédés de préparation de composés de pyridazine

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