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WO2025104224A1 - Variants de synthase d'edds - Google Patents

Variants de synthase d'edds Download PDF

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Publication number
WO2025104224A1
WO2025104224A1 PCT/EP2024/082456 EP2024082456W WO2025104224A1 WO 2025104224 A1 WO2025104224 A1 WO 2025104224A1 EP 2024082456 W EP2024082456 W EP 2024082456W WO 2025104224 A1 WO2025104224 A1 WO 2025104224A1
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edds
amino acid
group
synthase variant
synthase
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Inventor
Eva Mahler
Anna Christina Bohnenkamp
Christian Fleck
Marta Reinoso Garcia
Andrea Herold
Valerio FERRARIO
Stefan SEEMAYER
Srividhya SUNDARAM
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BASF SE
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BASF SE
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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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)

Definitions

  • the present invention provides new ethylenediamine-N,N'-disuccinic acid (EDDS) synthase variants. More specifically, new polypeptides having EDDS synthase activity, methods of using the EDDS synthases to produce EDDS, polynucleotides and vectors encoding the new EDDS synthase variants are provided.
  • Enzymes are protein-based biocatalysts widely used in a variety of different industries. They can be specific and enantioselective catalysts and can be employed under milder reaction conditions in comparison to traditional catalysts. They, for example, can be catalytically active at lower temperatures, which can result in an overall reduction of energy consumption of production processes. Additionally, enzymes are biodegradable and therefore increasingly used in various applications as sustainable alternative to petrochemistry.
  • Ethylenediamine-N,N'-disuccinic acid is a chelating agent that can bind metal ions in a stable chelator complex.
  • EDDS is biodegradable, has a low toxicity and is therefore an attractive alternative to traditional chelating agents such as ethylenediaminetetraacetic acid (EDTA).
  • EDTA is a synthetic chelator, currently widely used in industry, with very similar structure and properties to EDDS. However, EDTA is not biodegradable. For achieving a more sustainable industry, it is desired to use chelators, which are biodegradable.
  • EDDS is produced by reacting e.g. maleic acid or fumaric acid with ethylenediamine as described in US3158635A.
  • EP1757685A1 discloses EDDS synthases with specific substitution mutations.
  • the primary task of the present invention was thus to provide new EDDS synthase variants for the production of EDDS.
  • EDDS ethylenediamine-A/./V'-disuccinic acid synthase variant comprising one or more substitution at one or more amino acid position(s) selected from positions 7, 9, 18, 23, 29, 30, 37, 38, 39, 86, 87, 90, 91 , 115, 116, 119, 123, 160, 161 , 162, 235, 297, 325, 330, and 394 associated with substrate access, and/or at one or more amino acid position(s) selected from position(s) 10, 26, 27, 89, 159, 279, 282, 290, 294, 320, 322 and 323 associated with the active site, and/or at one or more amino acid position(s) selected from position(s) 58, 96, 120, 148, 156, 166, 181 , 297, 300, 317, 319, 359, 365, 371 and 448 associated with enzyme stability, of a parental polypeptide, wherein the parental poly
  • EDDS ethylenediamine-A
  • EDDS synthase describes a polypeptide or enzyme having EDDS synthase activity.
  • EDDS synthase variant refers to an enzyme, which is derived from a parental polypeptide and is thus a variant of this parental enzyme. It is preferred that the EDDS synthase variant exhibits an overall improved enzyme performance in comparison to the parental enzyme.
  • the numbering of the amino acid positions at which substitutions are present is preferably according to the numbering of the amino acid residues of the parental polypeptide.
  • the parental polypeptide and therefore also the basis for numbering is an enzyme having EDDS synthase activity according to SEQ ID NO.: 1 or 2, preferably of SEQ ID NO.: 1.
  • substitution in terms of the present invention relates to the change of an amino acid residue in comparison to the parental polypeptide. For example, if an isoleucine is present at position 166 of the parental polypeptide according to SEQ ID NO.: 1 and a serine is present in the EDDS synthase variant, this is called a substitution. Such a substitution is either described by the 3-letter amino acid code as lle166Ser or by the one letter code I166S. In describing the polypeptides of the present invention, the abbreviations for single amino acids are used according to the accepted IIIPAC single letter or three letter amino acid abbreviation is used.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at one or more amino acid position(s) associated with substrate access, in particular at one or more, preferably at two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or all amino acid positions selected from positions 7, 9, 18, 23, 29, 30, 37, 38, 39, 86, 87, 90, 91 , 115, 116, 119, 123, 160, 161 , 162, 235, 297, 325, 330, and 394.
  • substitutions at these positions contribute to an enhanced substrate access to the active site of the enzyme and thus to an overall enhanced performance of the enzyme.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at one or more amino acid position(s) associated with the active site, in particular at one or more, preferably two, three, four, five, six, seven, eight, nine, ten, eleven or all amino acid positions selected from positions 10, 26, 27, 89, 159, 279, 282, 290, 294, 320, 322 and 323.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at one or more amino acid position(s) associated with enzyme stability, in particular at one or more, preferably two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14 or all amino acid position(s) selected from position(s) 58, 96, 120, 148, 156, 166, 181 , 297, 300, 317, 319, 359, 365, 371 and 448. It was found in terms of the present invention that substitutions at these positions contribute to an improved stability of the enzyme and thus to an improved overall performance.
  • enzyme stability relates to the retention of enzymatic activity as a function of time during storage or operation. Retention of enzymatic activity as a function of time during storage is called “storage stability” and is preferred within the context of the present invention.
  • the enzyme of the invention shows an increased performance in view of an parental enzyme, wherein the parental enzyme is SEQ ID NO.: 1 or 2, and, wherein the EDDS synthase variant has a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to SEQ ID NO.: 1 or 2.
  • nucleic acid or amino acid sequences Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each other, these values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program or the EMBOSS Water Pairwise Sequence Alignments (polypeptide) program for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see Smith, T.F. & Waterman, M.S.
  • EMBOSS Water Pairwise Sequence Alignments amino acid sequences.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at amino acid position(s) associated with substrate access, preferably selected from positions 7, 9, 18, 23, 29, 30, 37, 38, 39, 86, 87, 90, 91 , 115, 116, 119, 123, 160, 161 , 162, 235, 297, 325, 330, and 394 and one or more substitution(s) at amino acid position(s) associated with the active site, preferably selected from position(s) 10, 26, 27, 89, 159, 279, 282, 290, 294, 320, 322 and 323.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at amino acid position(s) associated with substrate access, preferably selected from positions 7, 9, 18, 23, 29, 30, 37, 38, 39, 86, 87, 90, 91 , 115, 116, 119, 123, 160, 161 , 162, 235, 297, 325, 330, and 394 and one or more substitution(s) at amino acid position(s) associated with enzyme stability, preferably selected from position(s) 58, 96, 120, 148, 156, 166, 181 , 297, 300, 317, 319, 359, 365, 371 and 448.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at amino acid position(s) associated with active site, preferably selected from position(s) 10, 26, 27, 89, 159, 279, 282, 290, 294, 320, 322 and 323 and one or more substitution(s) at amino acid position(s) associated with enzyme stability, preferably selected from position(s) 58, 96, 120, 148, 156, 166, 181 , 297, 300, 317, 319, 359, 365, 371 and 448.
  • the EDDS synthase variant according to the present invention comprises one or more substitution(s) at amino acid position(s) associated with substrate access, preferably selected from positions 7, 9, 18, 23, 29, 30, 37, 38, 39, 86, 87, 90, 91 , 115, 116, 119, 123, 160, 161 , 162, 235, 297, 325, 330, and 394 and one or more substitution(s) at amino acid position(s) associated with the active site, preferably selected from position(s) 10, 26, 27, 89, 159, 279, 282, 290, 294, 320, 322 and 323 and one or more substitution(s) at amino acid position(s) associated with enzyme stability, preferably selected from position(s) 58, 96, 120, 148, 156, 166, 181 , 297, 300, 317, 319, 359, 365, 371 and 448.
  • the one or more amino acid substitution is an amino acid selected from the group consisting of polar, aliphatic, aromatic, positively charged and hydrophobic amino acids.
  • Polar amino acids are to be understood as amino acids having a polar side chain functional group.
  • polar amino acids are selected from serine, threonine, cysteine, asparagine, glutamine and tyrosine.
  • Aliphatic amino acids are to be understood as amino acids having an aliphatic side chain functional group.
  • aliphatic amino acids are selected from alanine, isoleucine, proline, valine and methionine.
  • Aromatic amino acids are to be understood as are to be understood as amino acids having an aromatic ring as functional group.
  • aromatic amino acids are selected from phenylalanine, tryptophan and tyrosine.
  • Positively charged amino acids are to the understood as amino acids having a positively charged sidechain as functional group at pH 7.
  • positively charged amino acids are selected from lysine, arginine and histidine.
  • Negatively charged amino acids are to the understood as amino acids having a negatively charged sidechain as functional group at pH 7.
  • negatively charged amino acids are selected from aspartic acid and glutamic acid.
  • Hydrophobic amino acids are to be understood as amino acids having a hydrophobic sidechain as functional group.
  • hydrophobic amino acids are selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan.
  • isoleucine is an amino acid having an aliphatic and hydrophobic functional side chain.
  • the substitution is an amino acid selected from the group consisting of alanine, aspartic acid, glutamine, lysine, leucine, isoleucine, methionine, threonine, tyrosine, serine, valine and glutamic acid.
  • the EDDS variant according to the present invention comprises one or more substitution(s) at amino acid position(s) selected from 9, 18, 23, 26, 120, 156, 166, 294, 322, 359 und 365.
  • the EDDS variant according to the present invention comprises one substitution at an amino acid position(s) selected from 9, 18, 23, 26, 120, 156, 166, 294, 322, 359 und 365.
  • the EDDS variant according to the present invention comprises two substitution at amino acid position(s) selected from 9 and 18, 9 and 23, 9 and 26, 9 and 120, 9 and 156, 9 and 166, 9 and 294, 9 and 322, 9 and 359, 9 and 365, 18 and 23, 18 and 26, 18 and 120, 18 and 156, 18 and 166, 18 and 294, 18 and 322, 18 and 359, 18 and 365, 23 and 26, 23 and 120, 23 and 156, 23 and 166, 23 and 294, 23 and 322, 23 and 359, 23 and 365, 26 and 120, 26 and 156, 26 and 166, 26 and 294, 26 and 322, 26 and 359, 26 and 365, 120 and 156, 26 and 166, 26 and 294, 26 and 322, 26 and 359, 26 and 365, 120 and 156, 120 and 166,
  • the EDDS variant according to the present invention comprises three substitution at amino acid position(s) selected from positions 9, 18 and 23, position 9, 23 and 26, positions 9, 26 and 120, positions 9, 120 and 156, positions 9, 156 and 166, positions 9, 166 and 294, positions 9, 294 and 322, positions 9, 322 and 359, positions 18, 23 and 26, positions 18, 26 and 120, positions 18, 120 and 156, positions 18, 156 and 166, positions 18, 166 and 294, positions 18, 294 and 322, positions 18, 322 and 359, positions 18, 359 and 365, positions 23, 26 and 120, positions 23, 120 and 156, positions 23, 256 and 166, positions 23, 166 and 294, positions 23, 294 and 322, positions 23, 322 and 359, positions 23, 359 and 465, positions 26, 120 and 156, positions 26, 156 and 166, positions 23, 166 and 294, positions 23, 294 and 322, positions 23, 322 and 359, positions 23, 359 and 365, positions 120, 156 and 166, positions 120, 166 and 294, positions 120, 294 and 322, positions 23,
  • substitution(s) is/are selected from the group consisting of D7M, T9L, T9I constructive T9S, T9E, T9Y, T9M, R10W, R10T, R10Y, R10S, R10K, K18T, Q23A, Y26D, D27E, D29G, A30S, S37D, A38E, V39T, V39I, V39F, A58G, A58K, R86D, R86N, R86S, R86H, T87V, T87I, T87L, L89M, L89A, L89T, P90A, P90N, P90S, Y91 F, Y91T, Y91 I, Y91V, Y91 L, K96I, K96T, K96V, L115M, L115E, L115Q, L115V, L115I, L115L, A116V, A116I, G119V, G119T, G119Q,
  • the one or more preferred substitution(s) associated with substrate access are D7M, T9L, T9I, T9S, T9E, T9Y, T9M, K18T, Q23A, D29G, A30S, S37D, A38E, V39T, V39I, V39F, R86D, R86N, R86S, R86H, T87V, T87I, T87L, P90A, P90N, P90S, Y91 F, Y91T, Y91 I, Y91V, Y91 L, L115M, L115E, L115Q, L115V, L115I, L115L, A116V, A116I, G119V, G119T, G119Q, G119A, G119D, F123Y, R160L, R160G, R160M, K161Q, K161 K, K161 R, K161V, K161 M, E162P, E162Q, E162R, E162
  • the one or more preferred substitution(s) associated with the active site are R10W, R10T, R10Y, R10S, R10K, Y26D, D27E, L89M, L89A, L89T, G159H, Q159L, Q159R, K297G, K297A, K297S, K297Q, I282A, I282T, I282L, I282M, D290V, D290I, D290L, R294L, R294Q, Y320L, Y320H, Y320N, Y320Y, Y320D, A322K, A322R, A322G, A322Q, A322V, A322I, A323D, A323V and A323E.
  • the one or more preferred substitution(s) associated with enzyme stability are A58G, A58K, K96I, K96T, K96V, K120E, G148Q, G148D, G148E, G148K, I166T, I166S, K181 R, K297G, K297A, K297S, K297Q, F300A, F300G, F300S, E317P, Y359A, Y359R, Y359D, Y359L, A365V, T371A M448A, M448G, and M448S.
  • the one or more substitution is/are selected from the group consisting of D7M, T9E, T9L, R10W, K18T, K18V, K18Q, Q23A, Q23L, Q23W, Y26I, Y26D, D27E, D29G, A30S, S37D, A38E, V39T, A58G, R86D, T87V, L89M, P90A, Y91 I, K96I, L115M, A116V, G119D, K120E, F123Y, G148Q, V156G, Q159H, R160W, K161 R, E162P, I166T, I166S, K181 R, W235R, T279G, I282M, D290L, R294L, R294Q, K297G, F300G, E317P, Q319T, Y320H, A322G, A322K, A323D, V325Q, R330
  • the one or more substitution is/are selected from the group consisting of T9E, K18V, K18Q, Q23L, Q23W, Y26I, V156G, I166S, I166T, K120E, R294Q, A322G, Y359A and S365V.
  • the EDDS synthase variant comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 3 to 63 or 78 to 87 or an amino acid sequence having at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 3 to 63 or 78 to 87.
  • the EDDS synthase variant comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 78 to 87 or an amino acid sequence having at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 78 to 87.
  • Another aspect of the present invention relates to a nucleic acid molecule encoding an EDDS synthase variant according to the invention.
  • the nucleic acid molecule is selected from the group consisting of SEQ ID NOs.: 66 to 77 or 88 to 97 or a nucleic acid sequence having at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 66 to 77 or 88 to 97.
  • Yet another aspect of the present invention relates to a vector, preferably a plasmid vector, comprising a sequence of a nucleic acid molecule according to the invention.
  • vector comprises any kind of construct suitable to carry polynucleotide sequences for transfer to a cell, or for stable or transient expression within a given cell.
  • This encompasses any kind of cloning vehicles, such as but not limited to plasmids, phagemids, viral vectors (e.g., phages), bacteriophage, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vectors specific for specific hosts of interest.
  • Foreign polynucleotide sequences usually comprise a coding sequence which may be referred to herein as “gene of interest”.
  • the gene of interest may comprise introns and exons, depending on the kind of origin or destination of host cell.
  • a vector according to the present invention herein may provide segments for transcription and translation of a polynucleotide upon transformation into a host cell or host cell organelles.
  • additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for its maintenance and/or replication in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc.
  • a vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g., plasmid or cosmid molecule).
  • suitable origins of replication include the f1 -ori and colE1.
  • the polynucleotide encoding the EDDS synthase of the present invention may be introduced into a vector by means of standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising a coding sequence may be suitable to be introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelles. A cloning vector suitable for expression of the polynucleotide sequence in the host cell or host cell organelles may be chosen.
  • a vector may replicate without integrating into the genome of a host cell, e.g., as a plasmid in a bacterial host cell, or it may integrate part of or all its DNA into the genome of the host cell and thus lead to replication and expression of its DNA.
  • Another aspect of the present invention relates to a genetically modified cell comprising the vector according to the invention and/or the nucleic acid molecule according to the invention for expressing an EDDS synthase variant according to the invention.
  • a polynucleotide encoding a polypeptide may be “expressed”.
  • expression or “gene expression” means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
  • the polynucleotide encoding the EDDS synthase as described herein may be transiently or stably introduced into a host cell and may be maintained nonintegrated, for example, as a plasmid.
  • stable transformation is due to integration of a nucleic acid comprising a recombinant coding sequence into the chromosomes or as an episome (separate piece of nuclear DNA).
  • transient transformation is due to a nucleic acid comprising a recombinant nucleic acid sequence not being integrated into the chromosomes or as an episome.
  • the introduction of a nucleic acid into a host cell may, for instance, but not limited thereto, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961 , Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742- 751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278).
  • Specific transformation protocols are known in the art for various types of host cells (see, e.g., for E. coli protoplast transformation see Hanahan, 1983, J. Mol. Biol. 166: 557-580).
  • Various host cells can be used for expressing the nucleic acid construct described herein.
  • Host cells comprising the genetic constructs described herein can be obtained by one of the methods described herein for introducing the polynucleotides into such host cells.
  • the host cell of the present invention does not naturally express the EDDS synthase.
  • the host cell is a recombinant host cell and the nucleic acid construct described herein is heterologous for the host cell.
  • the cell is selected from the group consisting of Escherichia coli, Bacillus licheniformis, Bacillus subitilis, Bacillus amyloliquefaciens, Saccharomyces cerevesiae, Komagataella phaffii, Hansenula polymorpha, Yarrowia lipolytica, Kluyveromyces lactis, Corynebacterium glutamicum, Pseudomonas putida, Vibrio natringens, Aspergillus niger and Basfia succiniproducens.
  • the host cell is a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell or a mammalian cell.
  • a method for producing EDDS comprising or consisting of the steps: i. cultivating at least one cell expressing an EDDS synthase variant according the invention, preferably a cell according to the invention and obtaining a whole cell biocatalyst; ii. optionally harvesting the obtained biocatalyst and/or purifying the expressed EDDS synthase variant; iii.
  • step vi providing a substrate selected from the group consisting of fumaric acid, fumarate, maleic acid, malic acid, maleic anhydrate, or a mixture thereof and ethylenediamine; iv. mixing the obtained biocatalyst of step i. or ii. with the educts provided in step iii.; v. incubating the mixture of step iv. at conditions suitable for the formation of EDDS, vi. obtaining a mixture containing EDDS, vii. optionally purifying the obtained mixture of step vi.
  • Step i. of the method according to the present invention includes culturing the at least one cell at conditions suitable for growth and expression of an EDDS synthases.
  • suitable culture conditions are known to the person skilled in the art and need to be adapted to the at least one cell to be cultured.
  • the at least one cell preferably does not naturally express the EDDS synthase.
  • the host cell is a recombinant host cell according to the invention and includes a nucleic acid molecule encoding an EDDS synthase variant according to the invention.
  • a nucleic acid molecule encoding an EDDS synthase variant according to the invention.
  • bi- or trivalent cations selected from the group consisting of alkaline earth metals, iron, zinc, copper, nickel, aluminium, titanium and manganese. The provided cations form a complex with the produced EDDS and thus shift the reaction equilibrium to the product.
  • Chelating agents such as EDDS and their respective alkali metal salts are useful sequestrants for metal ions (e. g. metal ions such as Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Fe3+).
  • metal ions e. g. metal ions such as Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Fe3+.
  • the EDDS synthesized using the EDDS synthase of the present invention is in the form of a metal-EDDS complex, preferably in the form of an Mg-EDDS complex.
  • the EDDS synthase variant of the present invention produces predominantly (S,S)-EDDS.
  • the EDDS synthase of the present invention produces (S,S)-EDDS with optical purity exceeding at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% enantiomeric excess (ee) or 100% (S,S)- EDDS.
  • step iv. is conducted by contacting the provided substrate and optionally the provided ions with the enzymes by e.g. providing them as part of one reaction mixture or contacting them via the use of a membrane and preferably under the use of a stirrer. Suitable mixing operations are known to the person skilled in the art.
  • step v. is conducted at conditions suitable for the formation of EDDS. Such conditions are known to the person skilled in the art, preferably those conditions as described herein.
  • the suitable conditions of step v. include: a temperature of between 20 °C and 60 °C, preferably of between 30°C and
  • the mixture obtained in step vi. is preferably further processed and the obtained EDDS is preferably purified in step vii. , preferably with a method selected from the group consisting of includes or is selected from the group consisting of filtration, crystallization, centrifugation, chromatography, precipitation, or a combination thereof.
  • purification refers to a process in which at least one component, e.g. the obtained EDDS, is separated from at least another component and transferred into a different compartment or phase, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier.
  • the EDDS obtained by any of the methods described herein can be used in a composition.
  • the composition can in principle be any composition.
  • the composition is a composition in which a chelating agent provides a beneficial effect.
  • EDDS produced by any of the methods described herein is used in the composition as builder.
  • compositions comprising EDDS obtained by any of the methods described herein include but are not limited to a detergent composition, a fertilizer composition, a pharmaceutical composition, home care compositions or personal care compositions, a nutritional supplement composition, a composition suitable for producing pulp or compositions used in the paper industry, in photographic supplies, for soil remediation, and in wastewater treatment.
  • the EDDS obtained by any of the methods described herein can be used in a detergent composition.
  • the detergent composition preferably is a laundry or hard surface cleaning detergent composition, suitable for home care and/or industrial and institutional (l&l) cleaning. Both the laundry and the hard surface cleaning composition can be in the form of a hand wash or an automated wash composition.
  • the hard surface cleaning detergent composition can preferably be a dish wash composition or a medical instrument cleaning composition.
  • the dish wash composition is an Automatic Dish Wash (ADW) composition.
  • ADW Automatic Dish Wash
  • Yet another aspect of the invention relates to the use of an EDDS synthase variant according to the invention for producing EDDS.
  • the invention is further characterized with illustrative, non-limiting examples.
  • SEQ ID NO.: 1 to 63 and 78 to 87 are amino acid sequences of EDDS synthase variants.
  • SEQ ID NO.: 64 to 77 and 88 to 97 are nucleic acid sequences encoding EDDS synthase variants.
  • DNA sequences encoding two EDDS synthases as control as well as EDDS synthase variants were generated using standard codon usage of Escherichia coli.
  • the DNA sequences were synthesized (BioCat GmbH) and cloned into the plasmid PDHE19.2 (Ress-Loeschke, M. et al., DE 19848129, 1998, (BASF AG)).
  • the gene of interest lies under control of a rhamnose inducible promoter (rhaBAD).
  • the resulting plasmids were used to transform competent cells (Chung, C.T. et al. , Proc Natl Acad Sci U S A, 1989, 86, 2172) of the E.
  • the E. coli strain TG10 is a rhaA' -derivate of E. coli TG1 (DSMZ 6056) transformed with pHSG575 (Takeshita, S. et al. , Gene, 1987, 61 , 63) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001 , 40, 397).
  • E. coli TG10 carrying the recombinant plasmids of the EDDS synthase EDDS01 and the EDDS synthase variants were used to inoculate 20 ml LB medium (Bertani, G., J Bacterial, 1951 , 62, 293) supplemented with 100 pg/ml ampicillin, 50 pg/ml spectinomycin, 20 pg/ml chloramphenicol, 12.5 pg/ml tetracyline, 0.2 mM isopropyl-B-D-thiogalactopyranosid, and 0.5 g/l rhamnose in a 100 ml baffled Erlenmeyer flask. The cultures were incubated at 37 °C for 18 h under shaking conditions (200 rpm).
  • Enzymatic activity was measured at 37 °C (results in table 2) or 50 °C (results in Table 3) by incubating 20 pl thawed cells expressing EDDS synthase EDDS01 and EDDS synthase variants (which were previously frozen at -20°C in the cultivation medium), normalized to OD 15, in 100 pl substrate buffer containing 71 ,6 g/l fumaric acid, 26,95 g/l magnesium hydroxide and 18.5 g/l ethylenediamine in 1 M Tris buffer at pH 8.5. 10 pl samples were taken 30 mins after starting the reaction.
  • the reaction was stopped by adding 490 pl stop/eluent solution (500 ml of 25% methanol, 1500 g water, 1 g/L copper II acetate-monohydrate, 2 g/L tetrabutylammoniumhydrate, pH 2,8 (adjusted with H3PO4)). Samples were then filtered through a 0.22 pm filter before being analyzed using HPLC analysis. For HPLC measurements the following settings were used: column: Aqua C18 (Phenomenex), temperature: 40 °C, UV detection at 254 nm, isocratic run with eluent solution.
  • volumetric enzymatic activity was calculated using the following formula:
  • delta c being the EDDS concentration produced in the time span delta t (here 0 to 30 min) and k as the dilution factor, which reflects the sample dilution.

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Abstract

La présente invention concerne de nouveaux variants de synthase d'acide éthylènediamine-N,N'-disuccinique (EDDS). Plus spécifiquement, l'invention concerne de nouveaux polypeptides ayant une activité de synthase d'EDDS ainsi que des procédés d'utilisation des synthases d'EDDS pour produire des EDDS, des polynucléotides et des vecteurs codant pour les nouveaux variants de synthase d'EDDS.
PCT/EP2024/082456 2023-11-17 2024-11-15 Variants de synthase d'edds Pending WO2025104224A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3158635A (en) 1959-03-18 1964-11-24 Stauffer Chemical Co Bis-adduction products and methods of preparing same
DE19848129A1 (de) 1998-10-19 2000-04-20 Basf Ag Verfahren zur Herstellung chiraler Carbonsäuren aus Nitrilen mit Hilfe einer Nitrilase oder Mikroorganismen, die ein Gen für die Nitrilase enthalten
WO2004050877A1 (fr) 2002-12-02 2004-06-17 Basf Aktiengesellschaft Systemes d'expression pouvant etre induits par le l-rhamnose
EP1757685A1 (fr) 2004-05-20 2007-02-28 Mitsubishi Rayon Co., Ltd. Acide ethylenediamine-n,n'-disuccinique:ethylenediamine lyase modifiee

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3158635A (en) 1959-03-18 1964-11-24 Stauffer Chemical Co Bis-adduction products and methods of preparing same
DE19848129A1 (de) 1998-10-19 2000-04-20 Basf Ag Verfahren zur Herstellung chiraler Carbonsäuren aus Nitrilen mit Hilfe einer Nitrilase oder Mikroorganismen, die ein Gen für die Nitrilase enthalten
WO2004050877A1 (fr) 2002-12-02 2004-06-17 Basf Aktiengesellschaft Systemes d'expression pouvant etre induits par le l-rhamnose
EP1757685A1 (fr) 2004-05-20 2007-02-28 Mitsubishi Rayon Co., Ltd. Acide ethylenediamine-n,n'-disuccinique:ethylenediamine lyase modifiee

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
BERTANI, G., J BACTERIOL, vol. 62, 1951, pages 293
CHANGCOHEN, MOLECULAR GENERAL GENETICS, vol. 168, 1979, pages 111 - 115
CHUNG, C.T. ET AL., PROC NATL ACAD SCI U S A, vol. 86, 1989, pages 2172
DATABASE Uniprot [online] 12 September 2018 (2018-09-12), GOEKER M.: "Argininosuccinate lyase from Chelatococcus asaccharovorans", XP093051582, Database accession no. A0A2V3UH70 *
DUBNAUDAVIDOFF-ABELSON, JOURNAL OF MOLECULAR BIOLOGY, vol. 56, 1971, pages 209 - 221
HANAHAN, J. MOL. BIOL., vol. 166, 1983, pages 557 - 580
JIELIN ZHANG ET AL: "Engineered C-N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, VERLAG CHEMIE, HOBOKEN, USA, vol. 59, no. 1, 19 November 2019 (2019-11-19), pages 429 - 435, XP072087747, ISSN: 1433-7851, DOI: 10.1002/ANIE.201910704 *
JIELIN ZHANG: "Supporting information to Engineered C-N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, 19 November 2019 (2019-11-19), XP093153587, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/10.1002/anie.201910704> DOI: 10.1002/anie.201910704 *
KOEHLERTHORNE, JOURNAL OF BACTERIOLOGY, vol. 169, 1987, pages 5271 - 5278
PROTEIN DATA BANK IN EUROPE: "PDB 6rx8 sequence < Protein Data Bank in Europe (PDBe) < EMBL-EBI", 30 October 2019 (2019-10-30), XP093153529, Retrieved from the Internet <URL:https://www.ebi.ac.uk/pdbe/entry/pdb/6rx8/protein/1> *
PROTEIN DATA BANK IN EUROPE: "PDB 6rx8 structure summary < Protein Data Bank in Europe (PDBe) < EMBL-EBI", 30 October 2019 (2019-10-30), XP093153486, Retrieved from the Internet <URL:https://www.ebi.ac.uk/pdbe/entry/pdb/6rx8> *
SHIGEKAWADOWER, BIOTECHNIQUES, vol. 6, 1988, pages 742 - 751
SMITH, T.F.WATERMAN, M.S.: "Identification of common molecular subsequences", JOURNAL OF MOLECULAR BIOLOGY, vol. 147, no. 1, 1981, pages 195 - 197, XP024015032, DOI: 10.1016/0022-2836(81)90087-5
TAKAHASHI ET AL.: "Production of (S,S)-Ethylenediamine-N,N'-disuccinic Acid from Ethylenediamine and Fumaric Acid by Bacteria", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 63, no. 7, 1999, pages 1269 - 1273, XP009033534, DOI: 10.1271/bbb.63.1269
TAKESHITA, S. ET AL., GENE, vol. 61, 1987, pages 63
TOMOYASU, T. ET AL., MOL. MICROBIOL., vol. 40, 2001, pages 397
WANG YILU ET AL: "Research progress on the biosynthesis and bioproduction of the biodegradable chelating agent (S,S)-EDDS", PROCESS BIOCHEMISTRY, ELSEVIER LTD, GB, vol. 116, 2 March 2022 (2022-03-02), pages 38 - 48, XP087015425, ISSN: 1359-5113, [retrieved on 20220302], DOI: 10.1016/J.PROCBIO.2022.02.025 *
YOUNGSPIZIZEN, JOURNAL OF BACTERIOLOGY, vol. 81, 1961, pages 823 - 829

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