WO2024028428A1 - Micro-organisme et procédé pour la production améliorée de sérine et/ou de cystéine - Google Patents

Micro-organisme et procédé pour la production améliorée de sérine et/ou de cystéine Download PDF

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WO2024028428A1
WO2024028428A1 PCT/EP2023/071522 EP2023071522W WO2024028428A1 WO 2024028428 A1 WO2024028428 A1 WO 2024028428A1 EP 2023071522 W EP2023071522 W EP 2023071522W WO 2024028428 A1 WO2024028428 A1 WO 2024028428A1
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microorganism
gene
serine
genes
cysteine
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Céline RAYNAUD
Laurence Dumon-Seignovert
Philippe Soucaille
Thomas Desfougeres
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Metabolic Explorer
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    • 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
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01017—L-Serine ammonia-lyase (4.3.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to a microorganism genetically modified for the improved production of serine and/or cysteine and to a method for the improved production of serine and/or cysteine using said microorganism.
  • Amino acids are used in many industrial fields, including the food, animal feed, cosmetics, pharmaceutical, and chemical industries and have an annual worldwide market growth rate of an estimated 5 to 7% (Leuchtenberger, et al., 2005).
  • serine and derivatives thereof as cysteine are particularly important for use in cosmetics and medical industry, in particular in pharmaceutics. Indeed, serine has been identified as one the most interesting biochemicals due to its potential use as a building block biochemical.
  • Serine and cysteine may be produced via chemical synthesis, or microbial fermentation. Due to the associated environmental advantages, replacement of chemical production by fermentation is considered as attractive and promising approaches. In addition, fermentation provides a useful way of using abundant, renewable, and/or inexpensive materials as the main source of carbon.
  • E. coli is a bacterial model organism for metabolic engineering, which is successfully employed as a cell factory for production of a range of biochemicals. The E. coli bacterium has long been used for the production of proteinogenic amino acids such as serine and cysteine, for many biotechnological applications.
  • Engineered strains which accumulates serine were constructed in the art, with different strategies to improve serine production. Some of which were mainly focusing on decreasing degradation of serine in the production organism. For example, genetically engineered microorganisms deficient in serine degradation pathways catalyzed by serine deaminases activity and serine hydroxymethyltransferase activity were disclosed in WO2016/120326. As a further example, others rather described stimulation of enzymes involved in the biosynthesis of serine as such, for example those leading to L-serine production from 3-phosphoglycerate as in US2019/233857. However, it is difficult to obtain optimal productions of serine, and consequently cysteine, because their biosynthesis competes with energy production and cell division for carbon utilization.
  • the present invention addresses the above needs, providing a microorganism genetically modified for the production of serine and/or cysteine and methods for the production of serine and/or cysteine using said microorganism.
  • the microorganism genetically modified for the production of serine and/or cysteine notably expresses a heterologous gapN gene coding an NADP-dependent glyceraldehyde- 3-phosphate dehydrogenase and has attenuated expression of gapA gene coding glyceraldehyde-3-phosphate dehydrogenase A and attenuated expression of sdaA and/or sdaB gene(s) coding L-serine deaminases (ie. L-serine dehydratases), as compared to an unmodified microorganism.
  • L-serine dehydratases L-serine dehydratases
  • the gapN gene codes an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase having at least 80% identity with GapN from Streptococcus mutans.
  • the gapA gene is deleted.
  • the microorganism further comprises an attenuation of the expression of the gapB and/or gapC genes as compared to an unmodified microorganism, preferably a deletion of the gapB and gapC genes.
  • the microorganism further comprises attenuation of the gpmA gene and/or glyA gene(s), as compared to an unmodified microorganism.
  • the microorganism further comprises an overexpression of at least one gene selected in the group consisting of: serA, serB, serC and eamA, preferably at least serA, more preferably at least serA and serB, as compared to an unmodified microorganism; where advantageously the gene serA is serA*.
  • the microorganism further comprises an overexpression of the gdhA gene as compared to an unmodified microorganism.
  • the microorganism further comprises the attenuation of at least one gene selected in the group consisting of tdcG and tdcB as compared to an unmodified microorganism.
  • the microorganism further comprises: a) an attenuation of the expression of the genes ptsHIcrr and/or the ptsG gene, preferably a deletion of the genes ptsHIcrr and/or the ptsG gene, b) an attenuation of the expression of the gpmM gene, preferably a deletion of the gpmM gene, and c) an overexpression of the galP gene, as compared to an unmodified microorganism.
  • the microorganism further comprises: a) an attenuation of the expression of the gpmM gene, preferably a deletion of the gpmM gene, and b) an overexpression of the esc genes, as compared to an unmodified microorganism.
  • the microorganism further comprises: a) an attenuation of the expression of the pykA and pykF genes, preferably a deletion of the pykA and pykF genes, and b) an overexpression of the scr genes, as compared to an unmodified microorganism.
  • the microorganism is genetically modified for the production of cysteine and comprises the overexpression of at least one gene selected in the group consisting of cysE, cysK and cysM, preferably the overexpression of cysE and cysK or cysE and cysM, more preferably of cysE and cysK, as compared to an unmodified microorganism; where advantageously the gene cysE is cysE*.
  • the expression of at least one gene selected from the group consisting of udhA, mgsA, ackA, ptA, pflAB, frdABCD, IdhA, adhE, zwf, edd, eda, gnd is attenuated, preferably the expression of the genes udhA, mgsA, zwf, edd, eda and gnd is attenuated, more preferably the expression of the genes udhA, mgsA, edd and eda is attenuated, as compared to an unmodified microorganism.
  • the microorganism belongs to the Escherichia genus, more preferably wherein the microorganism is Escherichia coli, the Corynebacterium genus, more preferably wherein the microorganism is Corynebacterium glutamicum, or the Streptococcus genus, more preferably wherein the microorganism is chosen among Streptococcus thermophilus and Streptococcus salivarius, most preferably wherein the microorganism is Escherichia coli.
  • the invention further relates to a method for the production of serine and/or cysteine comprising the steps of: a) culturing a microorganism genetically modified for the production of serine and/or cysteine as provided herein in an appropriate culture medium comprising a source of carbon, and b) recovering serine and/or cysteine from the culture medium.
  • the source of carbon is glucose, fructose, galactose, lactose, sucrose or any combination thereof.
  • step b) of the method comprises a step of purification of serine and/or cysteine.
  • a first aspect of the invention relates to a microorganism genetically modified for the production of serine and/or cysteine.
  • the term “microorganism,” as used herein, refers to a living microscopic organism, which may be a single cell or a multicellular organism and which can generally be found in nature.
  • the microorganism provided herein is preferably a bacterium.
  • the microorganism is selected within the Enterobacteriaceae, Streptococcaceae, or Corynebacteriaceae family. More preferably, the microorganism is a species of the Escherichia, Streptococcus, or Corynebacterium genus.
  • said Enterobacteriaceae bacterium is Escherichia coli
  • said Streptococcaceae bacterium is Streptococcus thermophilus or Streptococcus salivarius
  • said Corynebacteriaceae bacterium is Corynebacterium glutamicum.
  • the microorganism is Escherichia coli.
  • microorganism genetically modified
  • microorganism genetically modified refers to a microorganism or a strain of microorganism that has been genetically modified or genetically engineered. This means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is genetically modified when compared to the “parental” microorganism from which it is derived.
  • the “parental” microorganism may occur in nature (i.e., a wild-type microorganism) or may have been previously modified.
  • the recombinant microorganism of the invention may notably be modified by the introduction, deletion, and/or modification of genetic elements.
  • Such modifications can be performed, e.g., by genetic engineering or by adaptation, wherein a microorganism is cultured in conditions that apply a specific stress on the microorganism and induce mutagenesis and/or by forcing the development and evolution of metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure.
  • a microorganism genetically modified for the increased production of serine and/or cysteine means that said microorganism is a recombinant microorganism that has increased production of serine and/or cysteine as compared to a parent microorganism which does not comprise the genetic modification.
  • said microorganism has been genetically modified for increased production of serine and/or cysteine as compared to a corresponding unmodified microorganism.
  • a microorganism may notably be modified to modulate the expression level of an endogenous gene or the level of production of the corresponding protein or the activity of the corresponding enzyme.
  • endogenous gene means that the gene was present in the microorganism before any genetic modification. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace, endogenous regulatory elements. Endogenous genes may also be overexpressed by introducing one or more supplementary copies of the gene into the chromosome or on a plasmid. In this case, the endogenous gene initially present in the microorganism may be deleted.
  • Endogenous gene expression levels, protein production levels, or the activity of the encoded protein can also be increased or attenuated by introducing mutations into the coding sequence of a gene or into non-coding sequences. These mutations may be synonymous, when no modification in the corresponding amino acid occurs, or non- synonymous, when the corresponding amino acid is altered. Synonymous mutations do not have any impact on the function of translated proteins, but may have an impact on the regulation of the corresponding genes or even of other genes, if the mutated sequence is located in a binding site for a regulator factor. Non-synonymous mutations may have an impact on the function or activity of the translated protein as well as on regulation, depending the nature of the mutated sequence.
  • mutations in non-coding sequences may be located upstream of the coding sequence (i.e. , in the promoter region, in an enhancer, silencer, or insulator region, in a specific transcription factor binding site) or downstream of the coding sequence. Mutations introduced in the promoter region may be in the core promoter, proximal promoter or distal promoter.
  • Mutations may be introduced by site-directed mutagenesis using, for example, Polymerase Chain Reaction (PCR), by random mutagenesis techniques for example via mutagenic agents (Ultra-Violet rays or chemical agents like nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS)) or DNA shuffling or error-prone PCR or using culture conditions that apply a specific stress on the microorganism and induce mutagenesis.
  • mutagenic agents Ultra-Violet rays or chemical agents like nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS)
  • NVG nitrosoguanidine
  • EMS ethylmethanesulfonate
  • the insertion of one or more supplementary nucleotide(s) in the region located upstream of a gene can notably modulate gene expression.
  • a particular way of modulating endogenous gene expression is to exchange the endogenous promoter of a gene (e.g., wild-type promoter) with a stronger or weaker promoter to upregulate or downregulate expression of the endogenous gene.
  • the promoter may be endogenous (i.e., originating from the same species) or exogenous (i.e., originating from a different species). It is well within the ability of the person skilled in the art to select an appropriate promoter for modulating the expression of an endogenous gene.
  • Such a promoter be, for example, a Ptrc, Ptac, Ptet, or Plac promoter, or a lambda PL (PL) or lambda PR (PR) promoter.
  • the promoter may be “inducible” by a particular compound or by specific external conditions, such as temperature or light or a small molecule, such as an antibiotic.
  • a particular way of modulating endogenous protein activity is to introduce nonsynonymous mutations in the coding sequence of the corresponding gene, e.g., according to any of the methods described above.
  • a non-synonymous amino acid mutation that is present in a transcription factor may notably alter binding affinity of the transcription factor toward a cis-element, alter ligand binding to the transcription factor, etc.
  • a microorganism may also be genetically modified to express one or more exogenous or heterologous genes so as to overexpress the corresponding gene product (e.g., an enzyme).
  • an “exogenous” or “heterologous” gene as used herein refers to a gene encoding a protein or polypeptide that is introduced into a microorganism in which said gene does not naturally occur.
  • the gapN and Scr genes are notably heterologous genes in the context of the present invention.
  • a heterologous gene may be directly integrated into the chromosome of the microorganism, or be expressed extra-chromosomally within the microorganism by plasmids or vectors.
  • the heterologous gene(s) must be introduced into the microorganism with all of the regulatory elements necessary for their expression or be introduced into a microorganism that already comprises all of the regulatory elements necessary for their expression.
  • the genetic modification or transformation of microorganisms with one or more exogenous genes is a routine task for those skilled in the art.
  • One or more copies of a given heterologous gene can be introduced on a chromosome by methods well-known in the art, such as by genetic recombination.
  • a gene When a gene is expressed extra-chromosomally, it can be carried by a plasmid or a vector.
  • Different types of plasmid are notably available, which may differ in respect to their origin of replication and/or on their copy number in the cell.
  • a microorganism transformed by a plasmid can contain 1 to 5 copies of the plasmid, about 20 copies, or even up to 500 copies, depending on the nature of the selected plasmid.
  • Plasmids having different origins of replication and/or copy numbers are well-known in the art and can be easily selected by the skilled practitioner for such purposes, including, for example, pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pHS2, pPLc236, or PCL1920.
  • a heterologous gene encoding a protein of interest when expressed in a microorganism, such as E. coli, a synthetic version of this gene is preferably constructed by replacing non-preferred codons or less preferred codons with preferred codons of said microorganism which encode the same amino acid.
  • codon usage varies between microorganism species, and that this may impact the recombinant production level of a protein of interest.
  • codon optimization methods have been developed, and are extensively described by Graf eta/. (2000), Deml etal. (2001) and Davis & Olsen (2011).
  • the heterologous gene encoding a protein of interest is preferably codon-optimized for production in the chosen microorganism.
  • the heterologous gapN gene may be codon optimized for expression in a microorganism such as E. coli.
  • the skilled person is furthermore able to identify an appropriate polynucleotide coding for said polypeptide (e.g., in the available databases, such as Uniprot), or to synthesize the corresponding polypeptide or a polynucleotide coding for said polypeptide.
  • De novo synthesis of a polynucleotide can be performed, for example, by initially synthesizing individual nucleic acid oligonucleotides and hybridizing these with oligonucleotides complementary thereto, such that they form a double-stranded DNA molecule, and then ligating the individual double-stranded oligonucleotides such that the desired nucleic acid sequence is obtained.
  • production refer herein to an increase in the production level and/or activity of said protein in a microorganism, as compared to the corresponding parent microorganism that does not comprise the modification present in the genetically modified microorganism (i.e. , in the unmodified microorganism).
  • a heterologous gene or protein can be considered to be respectively “expressed” or “overexpressed” and “produced” or “overproduced” in a genetically modified microorganism when compared with a corresponding parent microorganism in which said heterologous gene or protein is absent.
  • the terms “attenuating” or “attenuation” of the synthesis of a protein of interest refer to a decrease in the production level and/or activity of said protein in a microorganism, as compared to the parent microorganism.
  • an “attenuation” of gene expression refers to a decrease in the level of gene expression as compared to the parent microorganism.
  • An attenuation of expression can notably be due to either the exchange of the wild-type promoter for a weaker natural or synthetic promoter or the use of an agent reducing gene expression, such as antisense RNA or interfering RNA (RNAi), and more particularly small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs).
  • Promoter exchange may notably be achieved by the technique of homologous recombination (Datsenko & Wanner, 2000).
  • the complete attenuation of the production level and/or activity of a protein of interest means that production and/or activity is abolished; thus, the production level of said protein is null.
  • the complete attenuation of the production level and/or activity of a protein of interest may be due to the complete suppression of the expression of a gene. This suppression can be either an inhibition of the expression of the gene, a deletion of all or part of the promoter region necessary for expression of the gene, or a deletion of all or part of the coding region of the gene.
  • a deleted gene can notably be replaced by a selection marker gene that facilitates the identification, isolation and purification of the modified microorganism.
  • suppression of gene expression may be achieved by the technique of homologous recombination, which is well-known to the person skilled in the art (Datsenko & Wanner, 2000).
  • Modulating the production level of one or more proteins may thus occur by altering the expression of one or more endogenous genes that encode said protein within the microorganism as described above and/or by introducing one or more heterologous genes that encode said protein(s) into the microorganism.
  • production level refers to the amount (e.g., relative amount, concentration) of a protein of interest (or of the gene encoding said protein) expressed in a microorganism, which is measurable by methods well-known in the art.
  • the level of gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like.
  • the level of production of the protein coded by said gene may be measured, for example by SDS-PAGE, HPLC, LC/MS and other quantitative proteomic techniques (Bantscheff et al., 2007), or, when antibodies against said protein are available, by Western Blot-lmmunoblot (Burnette, 1981), Enzyme-linked immunosorbent assay (e.g., ELISA) (Engvall and Perlman, 1971), protein immunoprecipitation, immunoelectrophoresis, and the like.
  • the copy number of an expressed gene can be quantified, for example, by restricting chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), qPCR, and the like.
  • Overexpression of a given gene or overproduction of the corresponding protein may be verified by comparing the expression level of said gene or the level of synthesis of said protein in the genetically modified organism to the expression level of the same gene or the level of synthesis of the same protein, respectively, in a control microorganism that does not have the genetic modification (i.e. , the parental strain or unmodified microorganism).
  • microorganism genetically modified for the production of serine and/or cysteine provided herein comprises
  • heterologous enzyme having NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity
  • the “activity” or “function” of an enzyme designates the reaction that is catalyzed by said enzyme for converting its corresponding substrate(s) into another molecule(s) (i.e., product(s)).
  • the activity of an enzyme may be assessed by measuring its catalytic efficiency and/or Michaelis constant. Such an assessment is described for example in Segel, 1993, in particular on pages 44-54 and 100-112, incorporated herein by reference.
  • the enzyme having NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity may be either a phosphorylating or a non-phosphorylating enzyme. It is preferably GapN. GapN may be of bacterial, archaeal, or eukaryotic origin. Preferably, GapN is of bacterial origin. GapN may notably be one of those described in Figure 4 of Iddar et al., 2005, incorporated herein by reference. In particular, the GapN enzyme may be from a species of the Streptococcus genus (e.g., from S. mutans, S. pyogenes), a species of the Bacillus genus (e.g., B.
  • Streptococcus genus e.g., from S. mutans, S. pyogenes
  • Bacillus genus e.g., B.
  • the GapN enzyme is from S. mutans, S. pyogenes, C. acetobutylicum, B. cereus, or P. sativum, more preferably from S. mutans.
  • GapN preferably has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the GapN enzyme having the sequence of SEQ ID NO: 23, 107, 109, 111 , or 113. More preferably, GapN has the sequence of SEQ ID NO: 23.
  • GapN may be a functional variant or functional fragment of one of the GapN enzymes described herein.
  • the corresponding gapN gene, which codes GapN preferably has at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 22, 106, 108, 110, or 112, more preferably SEQ ID NO: 22.
  • a “functional fragment” of an enzyme refers to parts of the amino acid sequence of an enzyme comprising at least all the regions essential for exhibiting the biological activity of said enzyme. These parts of sequences can be of various lengths, provided that the biological activity of the amino acid sequence of the enzyme of reference is retained by said parts. In other words, a functional fragment of an enzyme as provided herein is enzymatically active.
  • a “functional variant” as used herein refers to a protein that is structurally different from the amino acid sequence of a reference protein but that generally retains all the essential functional characteristics of said reference protein.
  • a variant of a protein may be a naturally-occurring variant or a non-naturally occurring variant.
  • Such non-naturally occurring variants of the reference protein can be made, for example, by mutagenesis techniques on the coding nucleic acids or genes, for example by random mutagenesis or site-directed mutagenesis.
  • Structural differences may be limited in such a way that the amino acid sequence of reference protein and the amino acid sequence of the variant may be closely similar overall, and identical in many regions. Structural differences may result from conservative or nonconservative amino acid substitutions, deletions and/or additions between the amino acid sequence of the reference protein and the variant. The only proviso is that, even if some amino acids are substituted, deleted and/or added, the biological activity of the amino acid sequence of the reference protein is retained by the variant. As a non-limiting example, such a variant of GapN conserves its NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity.
  • the capacity of the variants to exhibit such activity can be assessed according to in vitro tests known to the person skilled in the art. It should be noted that the activity of said variants may differ in efficiency as compared to the activity of the amino acid sequences of the enzymes of reference provided herein (e.g., the genes/enzymes provided herein of a particular species of microorganism or having particular sequences as provided in the corresponding SEQ ID NO).
  • a “functional variant” of an enzyme as described herein includes, but is not limited to, enzymes having amino acid sequences which are at least 60% similar or identical after alignment to the amino acid sequence encoding an enzyme as provided herein. According to the present invention, such a variant preferably has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence similarity or identity to the protein described herein. Said functional variant furthermore has the same enzymatic function as the enzyme provided herein. As a non-limiting example, a functional variant of GapN of SEQ ID NO: 23 has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to said sequence. As a non-limiting example, means of determining sequence identity are further provided below.
  • the attenuation of GapA activity and SdaA and/or SdaB activity results from an inhibition of expression of the gapA gene and the sdaA and/or sdaB gene(s) as compared to an unmodified microorganism.
  • the activity of the GapA enzyme and/or the activity of SdaA and/or SdaB enzyme(s) may be completely attenuated. Complete attenuation is preferably due to a partial or complete deletion of the gene coding for the enzyme.
  • GapA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 21.
  • the gapA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 20.
  • the gapA gene is deleted.
  • SdaA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 10.
  • the sdaA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 9.
  • SdaB has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 12.
  • the sdaB gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 11.
  • the microorganism genetically modified for the production of serine and/or cysteine microorganism of the present invention preferably comprises: - the expression of a heterologous gapN gene coding an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and
  • the genetically modified microorganism for production of serine and/or cysteine may comprise one or more additional modifications among those described below.
  • said microorganism may further comprise an attenuation of D-erythrose- 4-phosphate dehydrogenase (GapB) activity.
  • GapB D-erythrose- 4-phosphate dehydrogenase
  • production of GapB is partially or completely attenuated.
  • GapB has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 26.
  • attenuation of GapB activity results from an inhibition of the expression of the gapB gene coding said enzyme.
  • attenuation of expression results from a partial or complete deletion of the gapB gene.
  • the gapB gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 25.
  • Said microorganism may further comprise an attenuation of glyceraldehyde-3- phosphate dehydrogenase (GapC) activity.
  • GapC glyceraldehyde-3- phosphate dehydrogenase
  • production of GapC is partially or completely attenuated.
  • GapC has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 115, 117, or 119.
  • said attenuation results from an inhibition of expression of the gapC gene coding said enzyme.
  • attenuation of expression results from a partial or complete deletion of the gapC gene.
  • the gapC gene is a pseudogene.
  • gapC may refer to a functional gene or to a pseudogene.
  • the gapC pseudogene or functional gene preferably has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 27, 114, 116 or 118.
  • said pseudogene is advantageously deleted in order to avoid reversion of pseudogene into functional gene.
  • the microorganism comprises an attenuation of the expression of the gapB gene and deletion of gapC pseudogene as compared to an unmodified microorganism, more preferably a deletion of the gapB and gapC genes.
  • the microorganism genetically modified for the production of serine and/or cysteine preferably further comprises attenuated activity of the phosphoglycerate mutase GpmA, as compared to an unmodified microorganism.
  • GpmA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 29.
  • the attenuation of this phosphoglycerate mutase results from an attenuation of the gene coding said protein (i.e. the gene gpmA).
  • the gpmA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 28.
  • the microorganism genetically modified for the production of serine and/or cysteine preferably further comprises attenuated activity of the serine hydroxymethyltransferase GlyA, as compared to an unmodified microorganism.
  • GlyA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 121.
  • the attenuation of this serine hydroxymethyltransferase results from an attenuation of the gene coding said protein (i.e. the gene glyA).
  • the glyA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 120.
  • the microorganism comprises attenuation of the gpmA and/or glyA gene(s), as compared to an unmodified microorganism.
  • the microorganism for the production of serine may comprise an increased activity of at least one of the following L-serine deaminases: phosphoglycerate dehydrogenase (SerA), SerA*, phosphoserine phosphatase (SerB) and phosphoserine/phosphohydroxythreonine aminotransferase (SerC), as compared to an unmodified microorganism.
  • the microorganism for the production of serine may also comprise an increased activity of the cysteine/O-acetylserine exporter (EamA, also known as YdeD).
  • the microorganism for the production of serine comprises an overproduction of at least one of the following proteins: SerA, SerA*, SerB, SerC and EamA.
  • SerA* is a feedback resistant (FBR) protein.
  • feedback resistant protein refers to a protein which has been modified such that feedback inhibition of the protein (i.e., the reduction in enzyme activity mediated by the binding of the product to the enzyme) is reduced or even eliminated.
  • SerA, SerB, SerC and EamA have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NOs: 14, 16, 18 and 123, respectively.
  • SerA* is overproduced rather than SerA, said protein preferably has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 14.
  • SerA* comprises the substitution of asparagine residue at position 364 by an alanine residue when compared to SEQ ID NO: 14.
  • the overproduction of said one or more proteins results from an overexpression of at least one of the genes coding said protein (i.e., serA (or serA*), serB, serC and/or eamA genes).
  • serA, serB, serC and eamA genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 13, 15, 17 and 122, respectively.
  • serA* gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 13, wherein serA* codes for a protein having the substitution of asparagine residue at position 364 by an alanine residue with reference to the wild-type protein having the sequence SEQ ID NO: 14.
  • the microorganism is genetically modified for the production of serine and comprises an overexpression of the following genes: serA, serB, serC and eamA, and more preferably serA*, serB, serC and eamA, as compared to an unmodified microorganism.
  • overexpression of serA, serA*, serB, serC occurs by replacing the native promoter with an artificial promoter, such as the Ptrc promoter.
  • an artificial promoter such as the Ptrc promoter.
  • a vector comprising one or more genes under the control of a strong or inducible promoter e.g., the pCL1920 vector
  • the pCL1920 vector may be introduced into the microorganism and the gene(s) overexpressed.
  • eamA is overexpressed under the native promoter.
  • the microorganism for the production of serine may further comprise an increased activity of the glutamate dehydrogenase GdhA, as compared to an unmodified microorganism.
  • the microorganism for the production of serine comprises an overproduction of the glutamate dehydrogenase GdhA.
  • GdhA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 31 .
  • the overproduction of said protein results from an overexpression of the gene coding said protein (i.e., gdhA gene).
  • the gdhA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 30.
  • the microorganism is genetically modified for the production of serine and comprises an overexpression of the gdhA gene, as compared to an unmodified microorganism.
  • the microorganism genetically modified for the production of serine and/or cysteine preferably further comprises attenuated activity of at least one of the following L-serine deaminases: TdcG and TdcB, as compared to an unmodified microorganism.
  • TdcG and TdcB have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NOs: 36 and 38, respectively.
  • the attenuation of said one or more proteins results from an attenuation of the gene coding said protein (i.e., tdcG and/or tdcB genes).
  • the tdcG and tdcB genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 35 and 37, respectively.
  • the microorganism is genetically modified for the production of serine and/or cysteine and comprises an attenuation of at least one of the following genes: tdcG and tdcB genes, and more preferably of tdcG and tdcB genes, as compared to an unmodified microorganism.
  • the microorganism genetically modified for the production of serine or cysteine as described herein is further modified to be able to use glucose as a carbon source.
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above, further comprises: a) an attenuation of phosphoenolpyruvate-dependent phosphotransferase system (PTS) activity, preferably an inhibition of said activity, b) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably at least GpmM, and more preferably an inhibition of the GpmM activity, c) an increased galactose-proton symporter (GalP) activity, as compared to an unmodified microorganism.
  • PTS phosphoenolpyruvate-dependent phosphotransferase system
  • GpmM and/or GpmB preferably at least GpmM
  • GpmM galactose-proton symporter
  • the microorganism for the production of serine and/or cysteine comprises an attenuation of the proteins PtsHICrr and/or PtsG.
  • PtsH, Ptsl, and PtsG have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 42, 44, and 77, respectively.
  • Crr has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 46.
  • the attenuation of said one or more proteins results from an attenuation of the gene coding said protein (i.e. , ptsHIcrr and/or ptsG genes).
  • the ptsH, pstl, ptsG genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 41 , 43, and 76, respectively.
  • the err gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 45.
  • the microorganism is genetically modified for the production of serine an/or cysteine and comprises an attenuation of the ptsHIcrr and ptsG genes, as compared to an unmodified microorganism.
  • expression of PtsHICrr and/or PtsG is partially or completely attenuated.
  • attenuation of PtsHICrr and/or PtsG activity results from an inhibition of expression of the ptsHIcrr and/or ptsG genes coding said enzymes.
  • attenuation of expression results from a partial or complete deletion of the ptsHIcrr and/or ptsG genes, more preferably from a partial or complete deletion of the ptsHIcrr and ptsG genes.
  • the GpmM and GpmB proteins have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 40 and 79, respectively.
  • the microorganism for the production of serine and/or cysteine comprises an attenuation of the protein GpmM, as compared to an unmodified microorganism.
  • the attenuation of said one or more proteins results from an attenuation of the gene coding said protein (i.e., gpmM and/or gpmB genes).
  • the gpmM and gpmB genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 39 and 78, respectively.
  • the microorganism for the production of serine and/or cysteine comprises an attenuation of the gpmM gene, as compared to an unmodified microorganism.
  • GpmM and/or GpmB is partially or completely attenuated.
  • attenuation of GpmM and/or GpmB activity results from an inhibition of expression of the gpmM and/or gpmB genes coding said enzymes, most preferably from an inhibition of expression of the gpmM gene.
  • attenuation of expression results from a partial or complete deletion of the gpmM and/or gpmB genes, more preferably from a partial or complete deletion of the gpmM and/or gpmB genes, most preferably from a partial or complete deletion of the gpmM gene.
  • the microorganism for the production of serine and/or cysteine comprises an overproduction of the galactose-proton symporter GalP.
  • GalP has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 48.
  • the overproduction of said protein results from an overexpression of the gene coding said protein (i.e., galP gene), as compared to an unmodified microorganism.
  • the galP gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 47.
  • the microorganism is genetically modified for the production of serine and/or cysteine as described above and further comprises: a) an attenuation of the expression of the ptsHIcrr genes and/or the ptsG gene, preferably a deletion of the genes ptsHIcrr and/or the ptsG gene, b) an attenuation of the expression of the gpmM gene and/or the gpmB gene, preferably a deletion of the gpmM gene and/or the gpmB gene, and c) an overexpression of the galP gene, as compared to an unmodified microorganism.
  • the microorganism is further modified to comprise attenuation of glyA and/or overexpression of eamA, as compared to an unmodified microorganism, more preferably to comprise deletion of glyA and/or overexpression of eamA, and most preferably to comprise deletion of glyA and overexpression of eamA, as compared to an unmodified microorganism.
  • the microorganism genetically modified for the production of serine or cysteine as described herein is further modified to be able to use sucrose as a carbon source.
  • sucrose as a carbon source.
  • proteins involved in the import and metabolism of sucrose are overproduced.
  • the following proteins are overproduced:
  • ScrA Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and, said ScrK gene encodes ATP-dependent fructokinase, said ScrB sucrose 6- phosphate hydrolase (invertase), said ScrY sucrose porine, ScrR sucrose operon repressor.
  • genes coding for said proteins are overexpressed according to one of the methods provided herein.
  • the microorganism overexpresses:
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above, further comprises: a) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably at least GpmM, and more preferably an inhibition of the GpmM activity, b) an increased activity of Csc proteins involved in the import and metabolism of sucrose, as compared to an unmodified microorganism.
  • the GpmM and GpmB proteins have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 40 and 79, respectively.
  • the microorganism for the production of serine and/or cysteine comprises an attenuation of the protein GpmM, as compared to an unmodified microorganism.
  • the attenuation of said one or more proteins results from an attenuation of the gene coding said protein (i.e. , gpmM and/or gpmB genes).
  • the gpmM and gpmB genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 39 and 78, respectively.
  • the microorganism for the production of serine and/or cysteine comprises an attenuation of the gpmM gene, as compared to an unmodified microorganism.
  • GpmM and/or GpmB is partially or completely attenuated.
  • attenuation of GpmM and/or GpmB activity results from an inhibition of expression of the gpmM and/or gpmB genes coding said enzymes, most preferably from an inhibition of expression of the gpmM gene.
  • attenuation of expression results from a partial or complete deletion of the gpmM and/or gpmB genes, more preferably from a partial or complete deletion of the gpmM and/or gpmB genes, most preferably from a partial or complete deletion of the gpmM gene.
  • the microorganism for the production of serine and/or cysteine comprises an overproduction of the proteins CscBKAR.
  • the CscB sucrose permease, CscK fructokinase, CscA sucrose hydrolase, and CscR csc-specific repressor have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 51 , 53, 55 and 57, respectively.
  • the overproduction of said proteins results from an overexpression of the gene coding said protein (i.e., cscBKAR genes), as compared to an unmodified microorganism.
  • the cscBKAR genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 50, 52, 54 and 56, respectively.
  • the microorganism is genetically modified for the production of serine and/or cysteine as described above and further comprises: a) an attenuation of the expression of the gpmM gene and/or the gpmB gene, preferably a deletion of the gpmM gene and/or the gpmB gene, and b) an overexpression of the esc genes, as compared to an unmodified microorganism.
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above, further comprises: a) an attenuation of the pyruvate kinase activity, and more preferably an inhibition of the pyruvate kinase activity, b) an increased activity of Scr proteins involved in the import and metabolism of sucrose, as compared to an unmodified microorganism.
  • the PykA and PykF proteins have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 59 and 61 , respectively.
  • the attenuation of said proteins results from an attenuation of the gene coding said protein (i.e., pykA and pykF genes).
  • the pykA and pykF genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 58 and 60, respectively.
  • expression of PykA and PykF is partially or completely attenuated.
  • attenuation of PykA and PykF activity results from an inhibition of expression of the pykA and pykF genes coding said enzymes.
  • attenuation of expression results from a partial or complete deletion of the pykA and pykF genes, more preferably from a partial or complete deletion of the pykA and pykF genes.
  • the microorganism for the production of serine and/or cysteine comprises an overproduction of the proteins ScrKYABR.
  • the ScrK ATP-dependent fructokinase, the ScrY sucrose porine, the ScrA Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, the ScrB sucrose 6-phosphate hydrolase (invertase), and the ScrR sucrose operon repressor have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 63, 65, 67, 69 and 71 , respectively.
  • the overproduction of said proteins results from an overexpression of the gene coding said protein (i.e., scrKYABR genes), as compared to an unmodified microorganism.
  • the scrKYABR genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 62, 64, 66, 68 and 70, respectively.
  • the microorganism is genetically modified for the production of serine and/or cysteine as described above and further comprises: a) an attenuation of the expression of the pykA and pykF genes, preferably a deletion of the pykA and pykF genes, and b) an overexpression of the scr genes, as compared to an unmodified microorganism.
  • the microorganism is genetically modified for the production of serine and/or cysteine advantageously comprise a wild-type, not modified (not attenuated or deleted) GpmM activity or gpmM gene.
  • the microorganism is further modified to comprise attenuation of glyA and/or overexpression of eamA, as compared to an unmodified microorganism, more preferably to comprise deletion of glyA and/or overexpression of eamA, and most preferably to comprise deletion of glyA and overexpression of eamA, as compared to an unmodified microorganism.
  • the microorganism for the production of cysteine may comprise an increased activity of at least one of the following enzymes: serine acetyltransferase (CysE), cysteine synthase (CysK), and cysteine synthase (CysM), as compared to an unmodified microorganism.
  • the microorganism for the production of serine comprises an overproduction of at least one of the following proteins: CysE, CysE*, CysK and CysM.
  • CysE* is a feedback resistant (FBR) protein.
  • CysE, CysK and CysM have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NOs: 73, 75 and 81 , respectively.
  • said protein preferably has at least 80%, 90%, 95%, or 99,9% sequence similarity or sequence identity with the sequence of SEQ ID NO: 73.
  • CysE* comprises the substitution of threonine residue at position 167 by an alanine residue when compared to SEQ ID NO: 73.
  • the overproduction of said one or more proteins results from an overexpression of the gene coding said protein (i.e., cysE (or cysE *), cysK, and/or cysM genes).
  • the cysE, cysK, and cysM genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 72, 74 and 80, respectively.
  • the cysE* gene has at least 80%, 90%, 95%, or 99,9% sequence identity with the sequence of SEQ ID NO: 72, wherein cysE * codes for a protein having the substitution of threonine residue at position 167 by an alanine residue with reference to the wild-type protein having the sequence SEQ ID NO: 73.
  • the microorganism is genetically modified for the production of serine and comprises an overexpression of the genes cysE and cysK or of the genes cysE and cysM, more preferably of the genes cysE and cysK, and more preferably comprises an overexpression of the genes cysE* and cysK or of the genes cysE* and cysM, more preferably of the genes cysE* and cysK, as compared to an unmodified microorganism.
  • the microorganism is genetically modified for the production of serine and/or cysteine and further comprises:
  • soluble pyridine nucleotide transhydrogenase LldhA
  • MgsA methylglyoxal synthase
  • AckA acetyl-CoA carboxylase
  • PtA phosphate acetyltransferase
  • PflAB pyruvate formate lyase
  • FrdABCD lactate dehydrogenase
  • AdhE alcohol dehydrogenase
  • Zwf glucose-6-phosphate 1 -dehydrogenase
  • Zwf phosphogluconate dehydratase
  • Eda KHG/KDPG aldolase
  • 6-phosphogluconate dehydrogenase Gnd
  • LldhA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 8.
  • MgsA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 2.
  • AckA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 83.
  • PtA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 85.
  • PflA and PfIB have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 87 and 89, respectively.
  • FrdA, FrdB, FrdC, and FrdD have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 91 , 93, 95, and 97, respectively.
  • LdhA has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 99.
  • AdhE has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 101.
  • Zwf has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 103.
  • Edd has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 4.
  • Eda has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 6.
  • Gnd has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 105.
  • Attenuation of expression results from a partial or complete deletion of the gene encoding said protein (i.e. , at least one of the udhA, mgsA, ackA, ptA, pflAB, frdABCD, IdhA, adhE, zwf, edd, eda and gnd genes).
  • the udhA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 7.
  • the mgsA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 1 .
  • the ackA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 82.
  • the ptA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 84.
  • the pflAB genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 86 and 88, respectively.
  • the frdABCD genes have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 90, 92, 94, and 96, respectively.
  • the IdhA gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 98.
  • the adhE gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 100.
  • the zwf gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 102.
  • the edd gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 3.
  • the eda gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 5.
  • the gnd gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 104.
  • At least one gene selected from among udhA, mgsA, ackA, ptA, pflAB, frdABCD, IdhA, adhE, zwf, edd, eda and gnd is deleted.
  • the genes udhA, mgsA, zwf, edd, eda and gnd are attenuated as compared to an unmodified microorganism, more preferably deleted. More preferably, the genes udhA, mgsA, edd and eda are attenuated as compared to an unmodified microorganism, and most preferably deleted.
  • sequence identity is a function of the number of identical amino acid residues or nucleotides at positions shared by the sequences of said proteins.
  • sequence identity or “identity” as used herein in the context of two nucleotide or amino acid sequences more particularly refers to the residues in the two sequences that are identical when aligned for maximum correspondence.
  • percentage of sequence identity is used in reference to amino acid sequences, it is recognized that positions at which amino acids are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity).
  • percent identity between sequences may be adjusted upwards to correct for the conservative nature of the substitution.
  • sequence similarity or “similarity”.
  • sequence similarity is a function of the number of similar amino acid residues at positions shared by the sequences of said proteins.
  • the means of identifying similar sequences and their percent similarity or their percent identities are well-known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website.
  • sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN (http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.pl), with the default parameters indicated on those websites.
  • CLUSTALW http://www.ebi.ac.uk/clustalw/
  • MULTALIN http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.pl
  • sequence similarity and sequence identity between amino acid sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by a similar amino acid or by the same amino acid then the sequences are, respectively, similar or identical at that position. Sequence similarity may notably be expressed as the percent similarity of a given amino acid sequence to that of another amino acid sequence. This refers to the similarity between sequences on the basis of a “similarity score” that is obtained using a particular amino acid substitution matrix.
  • Sequence similarity may be calculated from the alignment of two sequences, and is based on a substitution score matrix and a gap penalty function.
  • the similarity score is determined using the BLOSUM62 matrix, a gap existence penalty of 10, and a gap extension penalty of 0.1 or the BLOSUM62 matrix, a gap existence penalty of 11 , and a gap extension penalty of 1.
  • no compositional adjustments are made to compensate for the amino acid compositions of the sequences being compared and no filters or masks (e.g., to mask off segments of the sequence having low compositional complexity) are applied when determining sequence similarity using web-based programs, such as BLAST.
  • the maximum similarity score obtainable for a given amino acid sequence is that obtained when comparing a sequence with itself.
  • the skilled person is able to determine such maximum similarity scores on the basis of the above-described parameters for any amino acid sequence.
  • a statistically relevant similarity can furthermore be indicated by a “bit score” as described, for example, in Durbin et al., Biological Sequence Analysis, Cambridge University Press (1998).
  • amino acid sequence can be optimally aligned as provided above, preferably using the BLOSUM62 matrix, a gap existence penalty of 10, and a gap extension penalty of 0.1.
  • Two sequences are “optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences.
  • a defined amino acid substitution matrix e.g., BLOSUM62
  • Percent similarity or percent identities as referred to herein are determined after optimal alignment of the sequences to be compared, which may therefore comprise one or more insertions, deletions, truncations and/or substitutions. This percent identity may be calculated by any sequence analysis method well-known to the person skilled in the art. The percent similarity or percent identity may be determined after global alignment of the sequences to be compared of the sequences taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970). Optimal alignment of sequences may preferably be conducted by the global alignment algorithm of Needleman and Wunsch (1970), by computerized implementations of this algorithm (such as CLUSTAL W) or by visual inspection.
  • sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software.
  • the parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4).
  • sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software.
  • the parameters used may notably be the following: “Gap open” equal to 10, “Gap extend” equal to 0.1 , and the BLOSUM62 matrix.
  • the percent similarity or identity as defined herein is determined via the global alignment of sequences compared over their entire length.
  • the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with the second amino acid sequence. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by a different but conserved amino acid residue, the molecules are similar at that position, and accorded a particular score (e.g., as provided in a given amino acid substitution matrix, discussed previously). When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position.
  • a particular score e.g., as provided in a given amino acid substitution matrix, discussed previously.
  • the percentage of sequence identity is calculated by comparing two optimally aligned sequences, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions and multiplying the result by 100 to yield the percentage of sequence identity.
  • PFAM protein family database of alignments and hidden Markov models; http://www.sanger.ac.uk/Software/Pfam/) represents a large collection of protein sequence alignments which may also be consulted by the skilled person. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs clusters of orthologous groups of proteins; http://www.ncbi.nlm.nih.gov/COG/
  • COGs clusters of orthologous groups of proteins; http://www.ncbi.nlm.nih.gov/COG/
  • Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the present invention relates to a method for the production of serine and/or cysteine using the microorganism described herein.
  • Said method comprises the steps of: a) culturing a microorganism genetically modified for the production of serine and/or cysteine as provided herein in an appropriate culture medium comprising a source of carbon, and b) recovering serine and/or cysteine from the culture medium.
  • the terms “fermentative process,” “fermentative production,” “fermentation,” or “culture” are used interchangeably to denote the growth of microorganism. This growth is generally conducted in fermenters with an appropriate growth medium adapted to the microorganism being used.
  • an “appropriate culture medium” or a “culture medium” refers to a culture medium optimized for the growth of the microorganism and the synthesis of serine or cysteine by the cells.
  • the culture medium e.g., a sterile, liquid media
  • the culture medium comprises nutrients essential or beneficial to the maintenance and/or growth of the microorganism such as carbon sources or carbon substrates, nitrogen sources; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts, for example magnesium salts, cobalt salts and/or manganese salts); as well as growth factors such as amino acids and vitamins.
  • the fermentation process is generally conducted in reactors with a synthetic, particularly inorganic, culture medium of known defined composition adapted to the microorganism, e.g., E. coli.
  • the inorganic culture medium can be of identical or similar composition to an M9 medium (Anderson, 1946), an M63 medium or a medium such as defined by Schaefer et al. (1999).
  • synthetic medium refers to a culture medium comprising a chemically defined composition on which organisms are grown.
  • source of carbon refers to any carbon source capable of being metabolized by a microorganism wherein the substrate contains at least one carbon atom.
  • said source of carbon is preferably at least one carbohydrate, and in some cases a mixture of at least two carbohydrates.
  • carbohydrate refers to any carbon source capable of being metabolized by a microorganism and containing at least three carbon atoms, two atoms of hydrogen.
  • the one or more carbohydrates may be selected from among the group consisting of: monosaccharides such as glucose, fructose, mannose, galactose, and the like, disaccharides such as sucrose, cellobiose, maltose, lactose, and the like, oligosaccharides such as raffinose, stacchyose, maltodextrins, and the like, polysaccharides such as cellulose, starch, or glycerol.
  • monosaccharides such as glucose, fructose, mannose, galactose, and the like
  • disaccharides such as sucrose, cellobiose, maltose, lactose, and the like
  • oligosaccharides such as raffinose, stacchyose, maltodextrins, and the like
  • polysaccharides such as cellulose, starch, or glycerol.
  • Preferred carbon sources are fructose, galactose, glucose, lactose, maltose, sucrose, or any combination thereof, more preferably glucose, fructose, galactose, lactose, and/or sucrose, most preferably glucose.
  • glucose fructose
  • galactose glucose
  • lactose maltose
  • sucrose sucrose
  • the method for the production of serine and/or cysteine comprises culturing the microorganism genetically modified as the first specific embodiment described above when the appropriate culture medium comprises glucose as a source of carbon.
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above further comprises: a) an attenuation of phosphoenolpyruvate-dependent phosphotransferase system (PTS) activity, preferably an inhibition of said activity, b) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably at least GpmM, and more preferably an inhibition of the GpmM activity, c) an increased galactose-proton symporter (GalP) activity, as compared to an unmodified microorganism.
  • PTS phosphoenolpyruvate-dependent phosphotransferase system
  • GpmM and/or GpmB preferably at least G
  • the method for the production of serine and/or cysteine comprises culturing the microorganism genetically modified as the second and third specific embodiments described above when the appropriate culture medium comprises sucrose as a source of carbon.
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above further comprises: a) an attenuation of the phosphoglycerate mutase GpmM and/or GpmB, preferably at least GpmM, and more preferably an inhibition of the GpmM activity, b) an increased activity of Csc proteins involved in the import and metabolism of sucrose, as compared to an unmodified microorganism.
  • the microorganism of the invention which is genetically modified for the production of serine and/or cysteine as described above, further comprises: a) an attenuation of the pyruvate kinase activity, and more preferably an inhibition of the pyruvate kinase activity, b) an increased activity of Scr proteins involved in the import and metabolism of sucrose, as compared to an unmodified microorganism.
  • the culture medium preferably comprises a nitrogen source capable of being used by the microorganism. Said source of nitrogen may be inorganic (e.g., (NH ⁇ SC i) or organic (e.g., urea or glutamate).
  • said source of nitrogen is in the form of ammonium or ammoniac.
  • said source of nitrogen is either an ammonium salt, such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium hydroxide and ammonium phosphate, or ammoniac gas, corn steep liquor, peptone (e.g., BactoTM peptone), yeast extract, meat extract, malt extract, urea, or glutamate, or any combination of two or more thereof.
  • an ammonium salt such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium hydroxide and ammonium phosphate, or ammoniac gas
  • corn steep liquor peptone (e.g., BactoTM peptone)
  • peptone e.g., BactoTM peptone
  • yeast extract e.g., BactoTM peptone
  • meat extract e.g., malt extract, urea, or glutamate
  • the nitrogen source may be derived from renewable biomass of microbial origin (such as beer yeast autolysate, waste yeast autolysate, baker's yeast, hydrolyzed waste cells, algae biomass), vegetal origin (such as cotton seed meal, soy peptone, soybean peptide, soy flour, soybean flour, soy molasses, rapeseed meal, peanut meal, wheat bran hydro lysate, rice bran and defatted rice bran, malt sprout, red lentil flour, black gram, bengal gram, green gram, bean flour, flour of pigeon pea, protamylasse) or animal origin (such as fish waste hydrolysate, fish protein hydrolysate, chicken feather; feather hydrolysate, meat and bone meal, silk worm larvae, silk fibroin powder, shrimp wastes, beef extract), or any other nitrogen containing waste. More preferably, said source of nitrogen is peptone and/or yeast extract.
  • vegetal origin such as cotton seed meal, soy peptone, soybean peptide
  • the person skilled in the art is able to define the culture conditions for the microorganisms according to the invention.
  • the bacteria are fermented at a temperature between 20°C and 55°C, preferably between 25°C and 40°C, more preferably between about 30°C to 39°C, even more preferably about 37°C.
  • a thermoinducible promoter is comprised in the microorganism provided herein, said microorganism is preferably fermented at about 39°C.
  • This process can be carried out either in a batch process, in a fed-batch process or in a continuous process. It can be carried out under aerobic, micro-aerobic or anaerobic conditions, or a combination thereof (for example, aerobic conditions followed by anaerobic conditions).
  • Under aerobic conditions means that oxygen is provided to the culture by dissolving the gas into the liquid phase. This could be obtained by (1) sparging oxygen containing gas (e.g., air) into the liquid phase or (2) shaking the vessel containing the culture medium in order to transfer the oxygen contained in the head space into the liquid phase.
  • oxygen containing gas e.g., air
  • the main advantage of the fermentation under aerobic conditions is that the presence of oxygen as an electron acceptor improves the capacity of the strain to produce more energy under the form of ATP for cellular processes. Therefore, the strain has its general metabolism improved.
  • Micro-aerobic conditions are defined as culture conditions wherein low percentages of oxygen (e.g., using a mixture of gas containing between 0.1 and 15% of oxygen, completed to 100% with inert gas such as nitrogen, helium or argon, etc.), is dissolved into the liquid phase.
  • low percentages of oxygen e.g., using a mixture of gas containing between 0.1 and 15% of oxygen, completed to 100% with inert gas such as nitrogen, helium or argon, etc.
  • Anaerobic conditions are defined as culture conditions wherein no oxygen is provided to the culture medium. Strictly anaerobic conditions are obtained by sparging an inert gas like nitrogen into the culture medium to remove traces of other gas. Nitrate can be used as an electron acceptor to improve ATP production by the strain and improve its metabolism.
  • step b) of the method comprises a step of filtration, ion exchange, crystallization, and/or distillation, more preferably a step of crystallization.
  • Serine or cysteine may be recovered from the culture medium and/or from the microorganism itself.
  • serine or cysteine is recovered from at least the culture medium.
  • serine may be purified by filtration or centrifugation for removing cells, by ion exchange and crystallization (at isoelectric point) (in particular as described in US3843441).
  • Cysteine may also be purified by filtration or centrifugation for removing cells, by ion exchange, concentration and crystallization (in particular as described in US8088949).
  • Protocol 1 Chrosomal modifications by homologous recombination, selection of recombinants and antibiotic cassette excision flanked by FRT sequences
  • protocol 2 Transduction of phage P1 used in this invention have been fully described in patent application WO2013/001055 (see in particular the “Examples Protocols” section and Examples 1 to 8, incorporated herein by reference).
  • Protocol 3 Construction of recombinant plasmids.
  • DNA fragments were PCR amplified using oligonucleotides (that the person skilled in the art will be able to define) and E. coli MG 1655 genomic DNA or an adequate synthetically synthesized fragment was used as a matrix.
  • the DNA fragments and chosen plasmid were digested with compatible restriction enzymes (that the person skilled in the art is able to define), then ligated and transformed into competent cells. Transformants were analysed and recombinant plasmids of interest were verified by DNA sequencing.
  • Protocol 4 Evaluation of L-Serine fermentation performance.
  • Production strains were evaluated in 500 mL Erlenmeyer baffled flasks using medium MMD (Table 1) for serine fermentation on dextrose adjusted to pH 6.8 or using medium MMS (Table 2) for serine fermentation on Sucrose.
  • a 10 mL preculture was grown at 37°C for 40 hours in a rich medium LB medium (10 g/L bactopeptone, 5 g/L yeast extract, 5 g/L NaCI). It was used to inoculate a 50 mL culture to an ODeoo of 0.2. When necessary, antibiotics were added to the medium (spectinomycin at a final concentration of 50 mg.L' 1 ). The temperature of the cultures was 37°C.
  • Serine yield (Yserine/dextrose) was expressed as followed:
  • Production strains were evaluated in 500 mL Erlenmeyer baffled flasks using medium MMD (Table 1) for cysteine fermentation on dextrose adjusted to pH 6.8 or using medium MMS (Table 2) for cysteine fermentation on Sucrose.
  • a 10 mL preculture was grown at 37°C for 40 hours in a rich medium LB medium (10 g/L bactopeptone, 5 g/L yeast extract, 5 g/L NaCI). It was used to inoculate a 50 mL culture to an ODeoo of 0.2. When necessary, antibiotics were added to the medium (spectinomycin at a final concentration of 50 mg.L' 1 ). The temperature of the cultures was 37°C.
  • Cysteine yield (Y C ysteine/dextrose) was expressed as followed: and Cysteine productivity (Pcysteine) was expressed as followed:
  • Cysteine yield (Y cy steine/sucrose) was expressed as followed: and Cysteine productivity (Pcysteine) was expressed as followed:
  • Biomass quantity variation is monitored using a spectrophotometer (Nicolet Evolution 100 UV-Vis, THERMO®).
  • the biomass production increases the turbidity of the growth medium. It is assayed by measuring the absorbance at a 600 nm wavelength. Each unit of absorbance corresponds to 2.2 x 10 9 +/- 2 x 10 8 cells/mL.
  • EXAMPLE 1 Strain constructions.
  • Serine producing strains Strains 1 to 14.
  • strain 1 was obtained by sequentially modifying the E. coli MG1655 strain by knocking out:
  • a serA* gene encoding a phosphoglycerate dehydrogenase SerA* protein havingthe substitution of asparagine residue at position 364 by an alanine residue with reference to the wild-type protein having the sequence SEQ ID NO: 14, and the serB gene encoding the phosphoserine phosphatase SerB protein (SEQ ID NO: 15 and 16, respectively) under their native promoter.
  • the serC gene encoding the phosphoserine I phosphohydroxythreonine aminotransferase SerC protein (SEQ ID NO: 17 and 18, respectively) under the trc promoter of SEQ ID NO: 19, leading to a construct Ptrc30/RBS01 -serC.
  • strain 2 was obtained by sequentially modifying strain 1 by knocking out the gapA gene encoding the glyceraldehyde-3-phosphate dehydrogenase A GapA protein (SEQ ID NO: 20 and 21 , respectively) and by overexpressing into the plasmid of strain 1 the heterologous gapN gene of Streptococcus mutans (SEQ ID NO: 22) encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase GapN protein (SEQ ID NO: 23, Uniprot Q59931) under the IPTG inducible trc promoter of SEQ ID NO: 24, thus leading to a construct Ptrc01/QP01/RBS09-gapNsm.
  • strain 3 was obtained by sequentially modifying strain 2 by knocking out the gapB gene encoding the D-erythrose-4-phosphate dehydrogenase GapB protein (SEQ ID NO: 25 and 26, respectively) and the gapC pseudogene (SEQ ID NO: 27) encoding the glyceraldehyde-3-phosphate dehydrogenase when this gene is intact,
  • strain 4 was obtained by modifying strain 3 by knocking out the gpmA gene encoding the 2,3-bisphosphoglycerate dependent phosphoglycerate mutase GpmA protein (SEQ ID NO: 28 and 29, respectively)
  • strain 5 was obtained by modifying strain 4 by overexpressing into the plasmid of strain 2 the gdhA gene encoding the glutamate dehydrogenase GdhA protein (SEQ ID NO: 30 and 31 , respectively) under the the PR promoter of SEQ ID NO: 32, thus leading to a construct PR-gdhA, with the cl857 allele from lambda phage (SEQ ID NO: 33 encoding the thermosensitive repressor protein of SEQ ID NO: 34) (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994).
  • strain 6 was obtained by modifying strain 5 by knocking out the tdcG gene encoding the L-serine deaminase III TdcG protein (SEQ ID NO: 35 and 36, respectively).
  • strain 7 was obtained by modifying strain 5 by knocking out the tdcB gene encoding the catabolic threonine dehydratase TdcB protein (SEQ ID NO: 37 and 38, respectively)
  • strain 8 was obtained by modifying strain 6 by knocking out the gpmM gene encoding the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase GpmM protein (SEQ ID NO: 39 and 40, respectively) and the ptsH-ptsl-crr operon (SEQ ID NO: 41 , 43 and 45, respectively) encoding the phosphocarrier protein HPr PtsH, the PTS enzyme I Ptsl and the Enzyme HA Glucose Crr proteins (SEQ ID NO: 42, 44 and 46, respectively), and by overexpressing the galP gene encoding the galactose: H+ symporter GalP protein (SEQ ID NO: 47 and 48, respectively) by replacing the native promoter by the trc promoter of SEQ ID NO: 49, thus leading to a construct Ptrc01/RBS01- galP.
  • strain 9 was obtained by modifying strain 7 by knocking out the gpmM gene encoding the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase GpmM protein (SEQ ID NO: 39 and 40, respectively) and the ptsH-ptsl-crr operon (SEQ ID NO: 41, 43 and 45, respectively) encoding the phosphocarrier protein HPr PtsH, the PTS enzyme I Ptsl and the Enzyme HA Glucose Crr proteins (SEQ ID NO: 42, 44 and 46, respectively), and by overexpressing the galP gene encoding the galactose: H+ symporter GalP protein (SEQ ID NO: 47 and 48, respectively) by replacing the native promoter by the trc promoter of SEQ ID NO: 49, thus leading to a construct Ptrc01/RBS01- galP.
  • strain 10 was obtained by modifying strain 1 by overexpressing into the plasmid of strain 1 the heterologous cscBKAR genes of E. coli EC3132 (SEQ ID NO: 50, 52, 54 and 56, respectively) encoding the sucrose permease CscB, sucrose fructokinase CscK, hydrolase CscA, and csc-specific repressor CscR (SEQ ID NO: 51, 53, 55 and 57, respectively) under the native promoter.
  • strain 11 was obtained by modifying strain 6 by knocking out the gpmM gene as described for strain 8 and by overexpressing into the plasmid of strain 5 the heterologous cscBKAR genes as described for strain 10.
  • strain 12 was obtained by modifying strain 7 by knocking out the gpmM gene as described for strain 8 and by overexpressing into the plasmid of strain 5 the heterologous cscBKAR genes as described for strain 10.
  • strain 13 was obtained by modifying strain 6 by knocking out the pykA gene encoding pyruvate kinase 2 PykA protein (SEQ ID NO: 58 and 59, respectively) and the pykF gene encoding pyruvate kinase 1 PykF protein (SEQ ID NO: 60 and 61 , respectively), and by overexpressing into the plasmid of strain 5 the heterologous scrKYABR genes of Salmonella sp.
  • strain 14 was obtained by modifying strain 7 by knocking out the pykA and pykF genes as described for strain 13 and by overexpressing into the plasmid of strain 5 the heterologous scrKYABR genes as described for strain 13.
  • Cysteine producing strains Strains 15 to 28.
  • strains 15 to 28 were obtained by sequentially modifying respectively strains 1 to 14.
  • Table 3 Biomass production, serine titer, productivity and yield for the different strains grown on the medium MMD described in Table 1.
  • the symbol “+” indicates an increase of a factor up to 2, the symbol “++” an increase by a factor between 2 and 3, and “+++” an increase by a factor greater than 4, as compared to the values of reference strain 1.
  • the symbol indicates a decrease of a factor up to 2, the symbol a decrease by a factor between 2 and 3, as compared to the values of reference strain 1.
  • gdhA gene in the strains 5 to 7 leads to an increase of both, final serine titer and productivity. It confirms the positive impact of increasing the glutamate availability into the bacterial cell.
  • Strains 8 and 9 exhibits a further improvement in serine production - specifically in the final serine titer and yield.
  • the suppression of genes coding enzymes consuming serine or serine precursors combined increases serine production.
  • Table 4 Biomass production, serine titer, productivity and yield for the different strains grown on the medium MMS described in Table 2.
  • the symbol “+” indicates an increase of a factor up to 2, the symbol “++” an increase by a factor between 2 and 3, and “+++” an increase by a factor greater than 4, as compared to the values of reference strain 7.
  • the symbol indicates a decrease of a factor up to 2, the symbol a decrease by a factor between 2 and 3, as compared to the values of reference strain 7.
  • strains 1 , 8 and 9 deals with the overexpression of the cscBKAR genes. They are coding for proteins conferring the ability of bacteria to passively import sucrose from outside of the cell. Strain 10 exhibits the same ability to grow and to produce serine, using sucrose as sole carbon source than the strain 1 when using dextrose.
  • Strains 13 and 14 contains the same metabolic pathway for the serine production except the absence of gpmM deletion but including the deletion of pyruvate kinase coding genes (pykA and pykF).
  • Table 5 Biomass production, cysteine titer, productivity and yield for the different strains grown on the medium MMD described in Table 1.
  • the symbol “+” indicates an increase of a factor up to 2, the symbol “++” an increase by a factor between 2 and 3, and “+++” an increase by a factor greater than 4, as compared to the values of reference strain 1.
  • the symbol indicates a decrease of a factor up to 2, the symbol a decrease by a factor between 2 and 3 as compared to the values of reference strain 1 .
  • gdhA gene in the strains 19 to 21 leads to an increase of both, final serine titer and productivity. It confirms the positive impact of increasing the glutamate availability into the bacterial cell.
  • Strains 22 and 23 exhibits a further improvement in cysteine production - specifically in the final cysteine titer and yield.
  • the suppression of genes coding enzymes consuming serine or serine precursors combined increases cysteine production.
  • Table 6 Biomass production, cysteine titer, productivity and yield for the different strains grown on the medium MMS described in Table 2.
  • the symbol “+” indicates an increase of a factor up to 2, the symbol “++” an increase by a factor between 2 and 3 and “+++” an increase by a factor greater than 4, as compared to the values of reference strain 7.
  • the symbol indicates a decrease of a factor up to 2, the symbol a decrease by a factor between 2 and 3, as compared to the values of reference strain 7.
  • strains 15, 22 and 23 to strains 24, 25 and 26 deals with the overexpression of the cscBKAR genes. They are coding for proteins conferring the ability of bacteria to passively import sucrose from outside of the cell. Strain 24 exhibits the same ability to grow and to produce cysteine, using sucrose as sole carbon source than the strain 15 when using dextrose.
  • strains 25 and 26 on sucrose are the same as those obtained using strain 22 and 23 on dextrose. These results demonstrate that changing the capacity to use different carbon source does not affect the cysteine production performances
  • Strains 27 and 28 contains the same metabolic pathway for the cysteine production except the absence of gpmM deletion but including the deletion of pyruvate kinase coding genes (pykA and pykF).
  • EXEMPLE 6 Serine production improvement by modifying glyA and/or eamA expression
  • strains 29 to 34 were obtained by knocking out the glyA gene encoding the serine hydroxymethyltransferase GlyA protein (SEQ ID NOs: 120 and 121 , respectively) and/or by overexpressing into the pCL1920-serA*-serB-serC vector described in example 1, the eamA gene encoding the cysteine/O-acetylserine exporter EamA protein (SEQ ID NOs: 122 and 123, respectively) under the native promoter.
  • Table 7 strains constructed.
  • Table 8 Biomass production, serine titer, productivity and yield for the different strains grown on the medium MMS described in Table 2.
  • the symbol “+” indicates an increase of a factor up to 2, the symbol “++” an increase by a factor between 2 and 3 and “+++” an increase by a factor greater than3, as compared to the values of reference strain 13.

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Abstract

La présente invention concerne un micro-organisme génétiquement modifié pour la production de sérine et/ou de cystéine, ledit micro-organisme comprenant l'expression d'un gène gapN hétérologue codant pour une glycéraldéhyde-3-phosphate déshydrogénase NADP-dépendante, et l'atténuation de l'expression du gène gapA et de l'expression du(des) gène(s) sdaA et/ou sdaB par rapport à un micro-organisme non modifié. La présente invention concerne également un procédé de production de sérine et/ou de cystéine à l'aide dudit micro-organisme.
PCT/EP2023/071522 2022-08-04 2023-08-03 Micro-organisme et procédé pour la production améliorée de sérine et/ou de cystéine WO2024028428A1 (fr)

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