Microorganisms and methods for improved valine production

Abstract

The present invention relates to a genetically modified microorganism for improved valine production, which overexpresses the ilvA gene encoding threonine deaminase and / or exhibits increased threonine deaminase activity and contains a mutant argP gene encoding a DNA-binding transcriptional dual regulator. The present invention also relates to a method for valine production using said microorganism.

Description

[Technical field]

[0001] The present invention relates to microorganisms genetically modified for improved valine production and methods for improved valine production using said microorganisms. [Background technology]

[0002] Amino acids are used in many industrial sectors, including the food, animal feed, cosmetics, pharmaceutical and chemical industries, with an estimated annual global market growth rate of 5-7% (Leuchtenberger et al., 2005).

[0003] Branched-chain amino acids also serve as precursors in the synthesis of herbicides and antibiotics, e.g., polyketides. Unlike many other amino acids that are metabolized in the liver, branched-chain amino acids are primarily metabolized in muscle and used as an energy source for physical activity.

[0004] Branched-chain amino acids (BCAAs) may be produced via chemical synthesis, extraction from protein hydrolysates, or microbial fermentation. Of these techniques, fermentation is the most commonly used today due to the associated economic and environmental benefits. In particular, fermentation offers a useful method to use abundant, renewable, and / or inexpensive materials as the primary carbon source. Furthermore, fermentation produces only the L-enantiomer, whereas when chemical synthesis is used, both D- and L-enantiomers are produced in equimolar amounts, necessitating further downstream isolation of the L-enantiomer. Summary of the Invention

[0005] Among these, the branched chain amino acid valine is particularly important for the nutrition of humans and many livestock species as it is among the nine essential amino acids that cannot be synthesized in mammals. L-valine has usually been produced by bacterial fermentation using mutant strains of Corynebacterium glutamicum and Escherichia coli. So far, most BCAA producing strains have been developed by random mutagenesis. These microbial strains have the disadvantage that it is difficult to further improve the strains because it is difficult to understand their exact physiological metabolism. Therefore, in the technical field to which the present invention belongs, there is a need to develop microorganisms with high ability of valine production, and as a result provide a novel method for producing valine at low cost.

[0006] The present invention relates to a microorganism genetically modified for valine production and a method for valine production using said microorganism. The microorganism genetically modified for valine production significantly overexpresses the ilvA gene encoding threonine deaminase and / or exhibits increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism. The microorganism also comprises a mutant argP gene encoding a DNA-binding transcriptional dual regulator.

[0007] Indeed, the inventors have surprisingly found that such microorganisms exhibit improved valine production by overexpressing the ilvA gene and / or by increasing the threonine deaminase activity, in contrast to prior art methods in which the expression of the ilvA gene or the corresponding enzymatic activity is not modified or is rather attenuated or even deleted.

[0008] Preferably, in a genetically modified microorganism according to the invention, the ilvA gene is overexpressed in the recombinant microorganism by, for example, modifying the promoter controlling the expression of the ilvA gene, by increasing the copy number of the ilvA gene present in the microorganism or by overexpressing the ilvA gene from a plasmid by improving the stability of ilvA mRNA or by increasing the amount of IlvA protein by optimizing the ribosome binding site, preferably by mutating the promoter controlling the expression of the ilvA gene.

[0009] More preferably, the ilvA gene is overexpressed in the recombinant microorganism by increasing the copy number of the ilvA gene present in the microorganism, resulting in two copies of the gene.

[0010] Preferably, the microorganism further comprises additional overexpression of the fepA gene encoding the ferric enterobactin outer membrane transporter and / or exhibits increased ferric enterobactin outer membrane transporter activity compared to the expression level and / or ferric enterobactin outer membrane transporter activity in the corresponding wild-type microorganism.

[0011] Preferably, the microorganism further comprises a deletion of at least one gene selected from the group consisting of IdhA, adhE and mgsA.

[0012] Preferably, the microorganism contains vdh, ilvD, ilvC, ilvB, and ilvN. * The method further comprises overexpression of at least one gene selected from the group consisting of:

[0013] Preferably, the microorganism belongs to the bacterial families Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcae, or Lactobacillae, or to the fungal, e.g., Hemiascomycetus, filamentous fungus, or yeast, families.

[0014] Preferably, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Bacillaceae bacterium is Bacillus subtilis, the Streptococcusceae bacterium is Streptococcus thermophiles, the Lactobacillaceae bacterium is Lactobacillus lactis, the hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, the filamentous fungus is Trichoderma rezeii or Aspergillus niger, and more preferably, the microorganism is Escherichia coli.

[0015] Preferably, the microorganism is the microorganism deposited on October 19, 2022 at the Collection Nationale de Cultures de Microorganismes, Pasteur Institute, 25 Rue du Docteur Roux, 75724 PARIS Cedex 15, FRANCE under the number CNCM I-5911.

[0016] The present invention further comprises: a) culturing a microorganism genetically modified for valine production according to any of the embodiments provided herein in an appropriate culture medium comprising a carbon source; b) recovering valine from the culture medium; The present invention further includes a method for producing valine, comprising:

[0017] Preferably, the step of recovering valine comprises at least a) clarification of the fermentation medium to remove insoluble organic impurities; b) a step of treatment of the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities; c) a step of evaporating water and crystallizing the product obtained; d) recovering valine; Includes.

[0018] Preferably, the carbon source is selected from arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose, or any polysaccharide, such as starch, cellulose or hemicellulose, and any combination thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Before describing the invention in detail, it is to be understood that the invention is not limited to the specifically exemplified microorganisms and / or methods, which may of course vary. It is also to be understood that the terminology used herein is merely for the purpose of describing certain embodiments of the invention, and is not intended to be limiting. The invention is limited only by the scope of the appended claims.

[0020] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.Furthermore, the practice of the present invention employs conventional microbiological and molecular biology techniques within the skill of the art, unless otherwise indicated.Such techniques are well known to those skilled in the art and are fully described in the literature.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, preferred materials and methods are provided.

[0022] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "microorganism" includes a plurality of such microorganisms, reference to an "endogenous gene" refers to one or more endogenous genes, and so forth.

[0023] The terms "comprise", "comprises" and "comprising" are used in the inclusive sense, i.e., used to specify the presence of stated features, but do not exclude the presence or addition of further features in various embodiments of the invention.

[0024] A first aspect of the present invention relates to a microorganism genetically modified for the production of valine.

[0025] The term "microorganism" as used herein refers to a living microscopic organism, which may be a single cell or a multicellular organism, and can generally be found in nature.In the context of the present invention, the microorganism is preferably a bacterium, yeast, or fungus.Preferably, the microorganism of the present invention is selected from the Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcus, or Lactobacillaceae family, or fungi, such as Hemiascomycetes, filamentous fungi, or yeast families.More preferably, the microorganism of the present invention is a species of Escherichia coli, Corynebacterium, Bacillus, Streptococcus, or Lactobacillus. Even more preferably, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Bacillaceae bacterium is Bacillus subtilis, the Streptococcusceae bacterium is Streptococcus thermophilus, the Lactobacillaceae bacterium is Lactobacillus lactis, the Hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, and the filamentous fungus is Trichoderma reesei or Aspergillus niger. Most preferably, the microorganism of the present invention is Escherichia coli.

[0026] The terms "recombinant microorganism" or "genetically modified microorganism" are used interchangeably herein and refer to genetically modified or engineered microorganisms or strains of microorganisms. This means that, according to the usual meaning of these terms, the microorganism of the invention is not found in nature and is genetically modified when compared to the "parent" microorganism from which it is derived. The "parent" microorganism may be naturally occurring (i.e., a wild-type microorganism) or may have been previously modified. The recombinant microorganism of the invention may be significantly modified by the introduction, deletion and / or modification of genetic elements. Such modifications may be performed, for example, by genetic engineering, by adaptation in which the microorganism is cultured under conditions that subject the microorganism to specific stresses to induce mutations, and / or by forcing the development and evolution of metabolic pathways by combining directed mutagenesis and evolution under specific selective pressures.

[0027] Microorganisms may be specifically modified to modulate the expression level of endogenous genes or the activity of the corresponding enzymes or transcription factors. The term "endogenous gene" means that the gene was present in the microorganism prior to any genetic modification. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to or to replace endogenous control elements. Endogenous gene expression levels, protein expression levels, or the activity of the encoded protein may also be increased or attenuated by introducing mutations in the coding or non-coding sequences of the gene. These mutations may be synonymous if no modification occurs in the corresponding amino acid, or non-synonymous if the corresponding amino acid is changed. Synonymous mutations do not have any effect on the function of the translated protein, but may affect the regulation of the corresponding gene or even the regulation of other genes if the mutated sequence is located in the binding site of a regulatory factor. Non-synonymous mutations may affect not only the function or activity of the translated protein, but also the regulation, depending on the nature of the mutated sequence.

[0028] In particular, mutations in non-coding sequences can be located upstream of the coding sequence (i.e. in promoter regions, enhancer, silencer, or insulator regions, specific transcription factor binding sites) or downstream of the coding sequence. Mutations introduced into the promoter region can be present in the core promoter, the proximal promoter, or the distal promoter. Mutations can be introduced by site-directed mutagenesis, for example using polymerase chain reaction (PCR), by random mutagenesis techniques, for example using mutagenic agents (UV light or chemical reagents such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS)), DNA shuffling, error-prone PCR, or using culture conditions that subject the microorganism to specific stresses to induce mutations. Gene expression can be specifically modulated by inserting one or more supplementary nucleotide(s) into a region located upstream of a gene.

[0029] A particular method of regulating the expression of an endogenous gene is to replace the endogenous promoter (e.g., wild-type promoter) of the gene with a stronger or weaker promoter to up-regulate or down-regulate the expression of the endogenous gene. The promoter can be endogenous (i.e., from the same species) or exogenous (i.e., from a different species). It is within the ability of a person skilled in the art to select a suitable promoter for regulating the expression of an endogenous gene. Such promoters may be, for example, Ptrc, Ptac, or Plac promoters, or PR or PL lambda promoters. The promoter can be "inducible" by a certain compound or by a certain external condition such as temperature or light.

[0030] A particular method of modulating endogenous protein activity is to introduce nonsynonymous mutations into the coding sequence of the corresponding gene, for example, according to any of the methods described above. Nonsynonymous amino acid mutations present in transcription factors can significantly alter the binding affinity of the transcription factor to cis elements and alter ligand binding to the transcription factor.

[0031] A microorganism may also be genetically modified to express one or more exogenous (i.e., heterologous) genes to express or overexpress the corresponding gene product (e.g., enzyme). As used herein, an "exogenous" or "heterologous" gene refers to a gene that encodes a protein or polypeptide that is introduced into a microorganism, in which the gene does not naturally occur. As used herein, a "heterologous gene" also refers to a gene that was endogenous to the microorganism (i.e., was present in the microorganism before any genetic modification), but that is not introduced into the location where the endogenous gene is / was located when introduced into the microorganism. More specifically, a heterologous gene may be an endogenous gene if the expression of the endogenous gene itself in the microorganism is reduced compared to the microorganism in which the gene naturally occurs (e.g., due to a mutation, complete or partial deletion of the gene, modification in the transcriptional control of the gene, etc.). In particular, the endogenous gene may no longer be expressed or may be expressed at a very low level. An exogenous gene may be directly integrated into the chromosome of the microorganism, or may be expressed extrachromosomally in the microorganism by a plasmid or vector. For successful expression, the exogenous gene(s) must be introduced into the microorganism together with all the control elements necessary for its expression or into a microorganism that already contains all the control elements necessary for its expression. Genetic modification or transformation of a microorganism with one or more exogenous genes is a routine procedure for those skilled in the art.

[0032] One or more copies of a given exogenous gene may be introduced onto a chromosome by methods well known in the art, for example, by genetic recombination. If a gene is expressed extrachromosomally, it may be carried by a plasmid or vector. Various types of plasmids are available, among others, and may differ with respect to origin of replication and / or copy number within the cell. For example, a microorganism transformed by a plasmid may contain 1-5 copies, about 20 copies, or even up to 500 copies of the plasmid, depending on the nature of the selected plasmid. Various plasmids with different origins of replication and / or copy numbers, including, for example, pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, or pPLc236, are well known in the art and may be easily selected by the skilled artisan for such purposes.

[0033] In the context of the present invention, it should be understood that when an exogenous gene encoding a protein of interest is expressed in a microorganism, a synthetic version of this gene can be preferably constructed by replacing unpreferred or less preferred codons with the preferred codons of said microorganism that code for the same amino acid. Indeed, it is well known in the art that codon usage differs between microbial species and that this can affect the recombinant expression level of the protein of interest. To overcome this problem, codon optimization methods have been developed and are extensively described by Graf et al. (2000), Deml et al. (2001) and Davis & Olsen (2011). Several software programs have been developed specifically to determine codon optimization, such as GeneOptimizer® software (Lifetechnologies) or OptimumGene™ software (GenScript). In other words, the exogenous gene encoding the protein of interest is preferably codon-optimized for expression in a microorganism.

[0034] The terms "express", "overexpress" or "overexpression" of a protein of interest, such as an enzyme, as used herein, refer to an increase in the expression level and / or activity of said protein in a microorganism compared to a corresponding parental microorganism that does not contain the modification(s) present in the genetically modified microorganism, also referred to as the corresponding wild-type microorganism. In some cases, the expression level may be similar to that of the parental microorganism. In other cases, the expression level may be superior to that of the parental microorganism. In cases where the parental microorganism does not contain the protein of interest, the terms "express" or "overexpression" refer to the presence of the protein of interest compared to its absence in the parental microorganism.

[0035] In contrast, the term "attenuate" or "attenuation" of a protein of interest refers to a reduction in the expression level and / or activity of said protein in a microorganism compared to the parent microorganism. Attenuation of expression can in particular be due to the replacement of the wild-type promoter with a weaker natural or synthetic promoter or the use of agents that reduce gene expression, such as antisense RNA or interfering RNA (RNAi), more particularly small interfering RNA (siRNA) or short hairpin RNA (shRNA). Promoter replacement can in particular be achieved by the technique of homologous recombination (Datsenko & Wanner, 2000). Complete attenuation of the expression level and / or activity of a protein of interest means that the expression and / or activity is abolished and thus the expression level of said protein is null. Complete attenuation of the expression level and / or activity of a protein of interest can be due to a 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 the expression of the gene, or a deletion of all or part of the coding region of the gene. The deleted genes may be replaced by selectable marker genes, which in particular facilitate the identification, isolation and purification of the modified microorganism. As a non-limiting example, suppression of gene expression can be achieved by the technique of homologous recombination, which is well known to those skilled in the art (Datsenko & Wanner, 2000).

[0036] Modulation of the expression level of one or more proteins can therefore occur by altering the expression of one or more endogenous genes encoding said proteins in the microorganism, as described above, or by introducing one or more heterologous genes encoding said proteins into the microorganism.

[0037] The term "expression level" as used herein refers to the amount (e.g., relative amount, concentration) of a protein of interest (or a gene encoding said protein) expressed in a microorganism, which may be measured by methods well known in the art. The level of gene expression may be measured by various known methods, including Northern blotting, quantitative RT-PCR, and the like. Alternatively, the expression level of the protein encoded by said gene may be measured, for example, by SDS-PAGE, HPLC, LC / MS, and other quantitative proteomic techniques (Bantscheff et al., 2007), or, if an antibody against said protein is available, by Western blot-immunoblot (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 may be quantified, for example, by Southern blotting using a probe based on the gene sequence after restriction of chromosomal DNA, fluorescent in situ hybridization (FISH), RT-qPCR, and the like.

[0038] Overexpression of a given gene or corresponding protein can be verified by comparing the expression level of said gene or protein in a genetically modified organism with the expression level of the same gene or protein in a control microorganism (i.e., the parent microorganism) that does not have the genetic modification.

[0039] The term "activity" or "function" as used herein in the context of an enzyme refers to the reaction catalyzed by said enzyme to convert the corresponding substrate(s) into another molecule(s) (i.e., product(s)). As is well known in the art, the activity of an enzyme can be assessed by measuring its catalytic efficiency and / or Michaelis constant. Such assessments are described, for example, in Segel, 1993, in particular pages 44-54 and 100-112, which are incorporated herein by reference.

[0040] The microorganism genetically modified for improved valine production provided herein overexpresses the ilvA gene encoding threonine deaminase, and / or shows increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism.Indeed, the inventors have surprisingly shown that the above genetic modifications significantly improve valine production in productivity and yield compared to parent microorganisms that do not contain these modifications.The improved valine production in this microorganism is particularly surprising because in the prior art methods involving the improvement of valine production, the ilvA gene expression or threonine deaminase activity is generally not modified or attenuated, or even deleted, i.e., null.

[0041] In the genetically modified microorganism for valine production provided by the present invention, any well-known prior art method can be used to overexpress ilvA gene expression or increase threonine deaminase activity. "Overexpression" or "overexpressing" is also used to indicate increasing transcription of a gene in a microorganism. Increasing transcription of a gene may be achieved by increasing the copy number of the gene and / or using a promoter that results in a higher level of expression of the gene.

[0042] For example, the ilvA gene can be overexpressed by overexpressing the ilvA gene from a plasmid, by modifying the promoter controlling the expression of the ilvA gene, by increasing the copy number of the ilvA gene present in the microorganism, or by increasing the amount of IlvA protein by improving the stability of ilvA mRNA or optimizing the ribosome binding site.

[0043] Preferably, expression of the ilvA gene is overexpressed, in particular by increasing the copy number of the ilvA gene present in the microorganism.

[0044] To increase the copy number of a gene in a microorganism, the gene is encoded chromosomally or extrachromosomally.If the gene is located on a chromosome, multiple copies of the gene can be introduced onto the chromosome by recombinant methods (including gene replacement) known to experts in the field.If the gene is located on an extrachromosomal site, as mentioned above, it can be carried by various types of plasmids that differ in terms of origin of replication and therefore copy number in the cell.

[0045] In a preferred embodiment, the copy number of the ilvA gene present in the genetically modified microorganism according to the present invention is at least two copies of the gene. Two, three, four or five copies of the gene are particularly preferred. Up to 10 or even 15 copies can also be considered. More preferably, two to five copies of the ilvA gene are present in the genetically modified microorganism according to the present invention. Most preferably, the ilvA gene is overexpressed in the genetically modified microorganism, resulting in two copies of the gene.

[0046] Increasing the translation of mRNA may be achieved by modifying the ribosome binding site (RBS). The RBS is a sequence on the mRNA that is bound by the ribosome when initiating the translation of a protein. This can be either the 5' cap of the mRNA in eukaryotes, the region 6-7 nucleotides upstream of the start codon AUG in prokaryotes (called the Shine-Dalgarno sequence), or the internal ribosome entry site (IRES) in viruses. By modifying this sequence, it is possible to change the rate of translation initiation of the protein, proportionally altering its production rate and controlling its activity in the cell. The same RBS sequence does not have the same effect due to the nature of the mRNA. By using the software RBS CALCULATOR (Salis, 2011), it is possible to optimize the strength of the RBS sequence to achieve the target translation initiation rate.

[0047] Improving mRNA stability may be achieved by reducing mRNA turnover and may be achieved by modifying the gene sequence in the 5'-untranslated region (5'-UTR) and / or the coding region, and / or the 3'-UTR (Carrier and Keasling, 1999).

[0048] In a preferred embodiment, the microorganism overexpresses the ilvA gene encoding the threonine deaminase of SEQ ID NO: 2, or a functional fragment or variant thereof.

[0049] As used herein, the term "functional fragment" of a reference protein having a biological activity of interest (e.g., an enzyme having threonine deaminase activity) refers to a portion of the amino acid sequence of the enzyme, said portion including at least all the essential regions for exhibiting the biological activity of said protein. These portions of the sequence may be of various lengths, provided that the biological activity of the reference amino acid sequence is retained by said portion. In other words, the functional fragments of the enzymes provided herein are enzymatically active.

[0050] A "functional variant" of an enzyme described herein (e.g., an enzyme having threonine deaminase activity) includes, but is not limited to, an enzyme having an amino acid sequence that is at least 60% identical after alignment with the amino acid sequence encoding the corresponding reference enzyme. According to the present invention, the variant preferably has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the protein described herein (e.g., IlvA protein). Thus, an enzyme having threonine deaminase activity preferably has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:2. More preferably, the gene encoding the enzyme having threonine deaminase activity has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 1. By way of non-limiting example, means for determining sequence identity are further provided below.

[0051] Increasing enzyme activity can also be achieved by improving protein catalytic efficiency, or by reducing protein turnover, or by reducing messenger RNA (mRNA) turnover, or by increasing gene transcription, or by increasing mRNA translation.

[0052] Improving the catalytic efficiency of a protein means increasing the kcat for a given substrate and / or for a given cofactor, and / or decreasing the Km, and / or increasing the Ki for a given inhibitor. kcat, Km and Ki are Michaelis-Menten constants that can be determined by the skilled artisan (Segel, 1993). Reducing the turnover of a protein means stabilizing the protein. Methods for improving the catalytic efficiency of a protein and / or reducing the turnover of a protein are well known to the skilled artisan. These include rational engineering by sequence and / or structure analysis and directed mutagenesis, as well as random mutagenesis and screening. Mutations can be introduced by site-directed mutagenesis by conventional methods such as polymerase chain reaction (PCR), or by random mutagenesis techniques such as the use of mutagenic agents (ultraviolet light, or chemical reagents such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS)) or the use of PCR techniques (DNA shuffling or error-prone PCR). Protein stabilization may also be achieved by adding a peptide sequence called a "tag" to either the N-terminus or C-terminus of the protein. Tags are well known to those skilled in the art. For example, glutathione-S-transferase (GST) can be used to stabilize proteins.

[0053] Genes and proteins are herein identified using the designation of the corresponding gene in E. coli (e.g., E. coli K12 MG1655 with Genbank accession number U00096.3) unless otherwise specified. However, in some cases, the use of these designations has a more general meaning according to the present invention and covers all of the corresponding genes and proteins in the microorganism. This is particularly the case for genes and proteins described herein that are not endogenous (i.e., heterologous) to the microorganism of the present invention, such as IlvA. As a specific example and as shown above, functional variants of IlvA, mutants and functional fragments thereof are also included herein. Specific embodiments are described in further detail below.

[0054] 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. Each PFAM makes it possible to visualize multiple alignments, identify protein domains, assess distribution among organisms, access other databases, and visualize known protein structures.

[0055] COGs (clusters of orthologous groups of proteins; http: / / www.ncbi.nlm.nih.gov / COG / ) are derived by comparing protein sequences from 43 fully sequenced genomes representing 30 major lineages. Each COG is defined from at least three lineages, allowing the identification of previously conserved domains.

[0056] Means for identifying similar sequences and their percent identity are well known to those skilled in the art and include, in particular, the BLAST program, which can be used with the default parameters indicated on the website http: / / www.ncbi.nlm.nih.gov / BLAST / . The sequences obtained can then be exploited using programs such as 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 these websites.

[0057] By using the reference information for known genes listed in GenBank, one skilled in the art can determine the equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine can be advantageously performed using consensus sequences, which can be determined by performing sequence alignments with genes from other microorganisms and designing degenerate probes to clone the corresponding genes in another organism. These routine methods of molecular biology are well known to those skilled in the art.

[0058] Sequence identity between amino acid sequences may be determined by comparing positions in each sequence that may be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same amino acid, then the sequences at that position are identical. The degree of sequence identity between proteins is a function of the number of identical amino acid residues at positions shared by the sequences of the proteins.

[0059] As a non-limiting example, to determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison. For example, gaps may be introduced into the sequence of the 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. If the position of the first sequence is occupied by the same amino acid residue as the corresponding position of the second sequence, the molecules are identical at that position.

[0060] The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences: % identity = number of identical positions / total number of overlapping positions x 100.

[0061] Optimal alignment of sequences can be performed by the global alignment algorithm of Needleman and Wunsch (1972), by computerized implementations of this algorithm (such as CLUSTAL W), or by visual inspection. The best alignment (i.e., the one that results in the highest percentage of identity between the compared sequences) produced by the various methods is selected.

[0062] In other words, the percentage of sequence identity is calculated by comparing two optimally aligned sequences, determining the number of positions where identical amino acids occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0063] The above definitions and preferred embodiments regarding functional fragments and functional variants of a protein apply mutatis mutandis to a nucleotide sequence, such as a gene, encoding a protein of interest (i.e. an enzyme having threonine deaminase activity).

[0064] In addition to the above-mentioned modifications, the genetically modified microorganism of the present invention may contain one or more additional modifications among the modifications described below. The modifications are advantageous because they can significantly further improve valine production, titer, and / or yield. The one or more modifications can significantly promote valine synthesis, suppress the use of valine as a substrate in downstream metabolic pathways, promote stable accumulation of valine, or suppress toxic accumulation of valine in the microorganism.

[0065] According to the present invention, the microorganism further comprises a mutated argP gene encoding a DNA-binding transcriptional dual regulator.

[0066] Preferably, the argP gene itself is mutated. More preferably, the argP gene is modified to result in an amino acid substitution at position 128 or the corresponding position. The most preferred embodiment in this regard is a substitution of Glu at position 128 with Asp (referred to herein as "argP* The argP gene is a transcription factor that, by binding directly to a promoter or to a control region, brings about various modulations (from overexpression to down-expression) of the expression of the target gene. Among the target genes, the following can be mentioned: gdhA, dapB, dapD, lysP, lysA, lysC, asd, dnaAN-recF, nrdAB-yfaE, argP, argO, argK, etc. Therefore, all modifications to the expression of the target gene caused by the substitution of an amino acid at position 128 of argP or the corresponding position should be considered to improve valine production and yield in a similar way compared to the usual modification in which argP is not mutated, i.e. wild type, and compared to the substitution of this same amino acid position in argP itself.

[0067] Corresponding positions can be determined by those skilled in the art, in particular using manual alignment or using alignment programs (e.g., BLASTP). Corresponding positions can also be based on structural alignment, for example, by using computer-simulated alignment of protein structures. The fact that amino acids of a polypeptide correspond to amino acids of a disclosed sequence means that standard alignment algorithms, such as the GAP algorithm, are used when the polypeptide and the disclosed sequence are aligned. In particular, corresponding amino acids can be identified when conserved amino acids are aligned such that the sequences have maximized identity or homology. As used herein, "at a corresponding position" refers to a position of interest (i.e., nucleotide base or amino acid residue number) in a nucleic acid molecule or protein relative to a position in a reference nucleic acid molecule or protein. A position of interest relative to a position in a reference protein can be, for example, an allelic variant, a heterologous protein, an amino acid sequence of the same protein of another species, etc. Corresponding positions can be determined by comparing and aligning sequences such that the number of paired nucleotides or amino acid residues is maximized. For example, the identity between the sequences may be greater than 95%, 96%, 97%, 98%, or more particularly greater than 99%. The position of interest is then given the number assigned to the sequence of the reference nucleic acid molecule or polypeptide.

[0068] Thus, according to the present invention, a microorganism overexpresses the ilvA gene encoding threonine deaminase in combination with a mutant argP gene encoding a DNA-binding transcriptional dual regulator as described above, and / or exhibits increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild type.

[0069] According to another preferred embodiment, the microorganism additionally overexpresses the fepA gene encoding the ferric enterobactin outer membrane transporter and / or exhibits increased ferric enterobactin outer membrane transporter activity compared to the expression level and / or ferric enterobactin outer membrane transporter activity in the corresponding wild-type microorganism.

[0070] As described herein, any method known in the art may be used to overexpress the fepA gene in the microorganisms described in the present invention. Thus, any technical means that results in increased fepA gene expression or increased ferric enterobactin outer membrane transporter activity compared to the expression level and / or ferric enterobactin outer membrane transporter activity in the corresponding wild-type microorganism may be used to genetically modify a microorganism in the context of the present invention. In particular, and by way of example only, by genetic modification, for example by modifying the promoter controlling the expression of the fepA gene, by increasing the copy number of the fepA gene present in the microorganism, or by overexpressing the fepA gene from a plasmid by improving the stability of fepA mRNA or by optimizing the ribosome binding site to increase the amount of FepA protein, preferably by mutating the promoter controlling the expression of the fepA gene. Indeed, any modification that may result in increased fepA gene expression or increased ferric enterobactin outer membrane transporter activity compared to the expression level and / or ferric enterobactin outer membrane transporter activity in the corresponding wild-type microorganism is included in the scope of the present invention.

[0071] Preferably, the microorganism further comprises an increased expression of the fepA gene by at least one base substitution in the promoter sequence controlling the expression of the fepA gene. More preferably, the at least one base substitution, in particular the one base substitution, is located about 10 pairs of nucleotides upstream from the transcription start point, and is immediately downstream of the "-10 box" (also called "Pribnow box"), which is composed of 6 nucleotides, optionally with some variations, but generally a TATAAT sequence. In particular, the one nucleotide substitution is made 3 nucleotides upstream from the transcription start of the promoter controlling the expression of the fepA gene as set forth in SEQ ID NO:7. More preferably, the thymine nucleotide at position -3 from the transcription start of the fepA promoter is replaced with a cytosine nucleotide as set forth in SEQ ID NO:8.

[0072] In another advantageous embodiment of the invention, the microorganism overexpresses the ilvA gene encoding threonine deaminase in combination with overexpression of the fepA gene encoding the iron enterobactin outer membrane transporter as described above and / or increased iron enterobactin outer membrane transporter activity, and / or exhibits increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism.

[0073] In a further advantageous embodiment of the invention, the microorganism overexpresses the ilvA gene encoding threonine deaminase in combination with a mutant argP gene encoding a DNA-binding transcriptional dual regulator as described above and in combination with overexpression of the fepA gene encoding the ferric enterobactin outer membrane transporter as described above and / or increased ferric enterobactin outer membrane transporter activity, and / or exhibits increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism.

[0074] According to yet another preferred embodiment, the microorganism further comprises attenuated expression of one or more of the following proteins: lactate dehydrogenase (LdhA), alcohol dehydrogenase (AdhE), methylglyoxal synthase (MgsA), fumarate reductase complex (FrdABCD), pyruvate formate lyase (PflAB), acetate kinase (AckA) and phosphate acetyltransferase (Pta), and / or branched chain amino acid transporter (BrnQ and LivKHMGF), said genes being endogenous, in particular in E. coli.

[0075] Preferably, LdhA has at least 80%, 90%, 95% or 100% sequence similarity or identity with the sequence of SEQ ID NO: 10. Preferably, AdhE has at least 80%, 90%, 95% or 100% sequence similarity or identity with the sequence of SEQ ID NO: 12. Preferably, MgsA has at least 80%, 90%, 95% or 100% sequence similarity or identity with the sequence of SEQ ID NO: 14. Preferably, FrdA, FrdB, FrdC and FrdD have at least 80%, 90%, 95% or 100% sequence similarity or identity with the sequences of SEQ ID NO: 16, 18, 20 and 22, respectively. Preferably, PflA and PflB have at least 80%, 90%, 95% or 100% sequence similarity or identity with the sequences of SEQ ID NO: 24 and 26, respectively. Preferably, AckA has at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequence of SEQ ID NO: 28. Preferably, Pta has at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequence of SEQ ID NO: 30. Preferably, BrnQ has at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequence of SEQ ID NO: 32. Preferably, LivK, LivH, LivM, LivG and LivF have at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequences of SEQ ID NO: 34, 36, 38, 40 and 42, respectively.

[0076] Preferably, the attenuation of expression results from a partial or complete deletion of the genes encoding said proteins (i.e., the IdhA, adhE, mgsA, frdABCD, pflAB, ackA-pta, brnQ and / or livKHMGF genes). Preferably, the genetically modified microorganism of the present invention further comprises a deletion of at least one gene selected from the group consisting of IdhA, adhE, and mgsA.

[0077] Preferably, the IdhA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 9. Preferably, the adhE gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 11. Preferably, the mgsA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 13. Preferably, the frdABCD genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 15, 17, 19 and 21, respectively. Preferably, the pflAB genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 23 and 25, respectively. Preferably, the ackA-pta gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 27 and 29, respectively. Preferably, the brnQ gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 31. Preferably, the livKHMGF gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 33, 35, 37, 39, and 41, respectively.

[0078] Valine-producing microorganisms express the following proteins: ketol-acid reductoisomerase (NADP(+)) (IlvC), dihydroxyacid dehydratase (IlvD), acetolactate synthase (IlvBN *), valine dehydrogenase (Vdh), branched chain amino acid aminotransferase (IlvE), and L-valine exporter (YgaZH). Preferably, the dehydrogenase is leucine or valine dehydrogenase.

[0079] Preferably, IlvC has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 44. Preferably, IlvD has at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 46. Preferably, IlvB and IlvN * have at least 80%, 90%, 95%, or 100% sequence similarity or sequence identity with the sequences of SEQ ID NOs: 48 and 52, respectively, and IlvN * contains substitutions G20D, V21D and M22F where the sequence is not 100% identical to SEQ ID NO: 52. Preferably, Vdh has at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequence of SEQ ID NO: 54. Preferably, IlvE has at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequence of SEQ ID NO: 57. Preferably, YgaZ and YgaH have at least 80%, 90%, 95% or 100% sequence similarity or identity to the sequences of SEQ ID NO: 59 and 61, respectively.

[0080] Preferably, the overexpression of one or more of said proteins is by regulating the genes encoding said proteins (i.e., ilvC, ilvD and / or ilvBN * Preferably, the genetically modified microorganism of the present invention is characterized in that it results from overexpression of the vdh, ilvD, ilvC, ilvB, and ilvN genes. * The method further comprises overexpression of at least one gene selected from the group consisting of:

[0081] Preferably, the ilvC gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 43. Preferably, the ilvD gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 45. Preferably, the ilvB and ilvN genes * The gene has at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NOs: 47 and 51, respectively, and is * The genes encode amino acids with substitutions G20D, V21D, and M22F with reference to the wild-type protein having the sequence of SEQ ID NO: 50. Preferably, vdh has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 53. Preferably, ilvE has at least 80%, 90%, 95%, or 100% sequence identity with the sequence of SEQ ID NO: 56. Preferably, ygaZ and ygaH have at least 80%, 90%, 95%, or 100% sequence identity with the sequences of SEQ ID NO: 58 and 60, respectively.

[0082] As stated above, the microorganism of the present invention may belong to the bacterial, fungal or yeast family.

[0083] Preferably, said microorganism belongs to the family Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcaceae or Lactobacillaceae, or to a family of fungi, such as hemisocomycetes, filamentous fungi or yeasts.

[0084] According to a preferred embodiment, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Bacillaceae bacterium is Bacillus subtilis, the Streptococcusceae bacterium is Streptococcus thermophilus, the Lactobacillaceae bacterium is Lactobacillus lactis, the hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, the filamentous fungus is Trichoderma reesei or Aspergillus niger, and more preferably, the microorganism is Escherichia coli.

[0085] Also, the microorganism according to the present invention may be genetically modified to contain either a modified endogenous gene / enzyme or a heterologous gene / enzyme. Preferably, the microorganism contains an endogenous gene or enzyme having threonine deaminase activity, more preferably the endogenous ilvA gene encoding threonine deaminase.

[0086] In a further embodiment, if the microorganism described herein is unable to use sucrose as a carbon source, said microorganism is modified to be able to use sucrose as a carbon source.Preferably, proteins involved in sucrose uptake and metabolism are overexpressed.Preferably, the following proteins are overexpressed: - CscB sucrose permease (SEQ ID NO: 63), CscA sucrose hydrolase (SEQ ID NO: 65), CscK fructokinase (SEQ ID NO: 67) and CscR csc-specific repressor (SEQ ID NO: 69), or - ScrA enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (SEQ ID NO: 75), the ScrK gene encoding an ATP-dependent fructokinase (SEQ ID NO: 71), ScrB sucrose 6-phosphate hydrolase (invertase) (SEQ ID NO: 77), ScrY sucrose porin (SEQ ID NO: 73), and ScrR sucrose operon repressor (SEQ ID NO: 79).

[0087] Preferably, the gene encoding said protein is overexpressed according to one of the methods provided herein. Preferably, the E. coli microorganism overexpresses: - a heterologous cscBKAR gene of E. coli EC3132 (cscB gene SEQ ID NO: 62; cscK gene SEQ ID NO: 66; cscA gene SEQ ID NO: 64, and cscR gene SEQ ID NO: 68), or - heterologous scrKYABR genes of the genus Salmonella (scrK gene SEQ ID NO:70; scrY gene SEQ ID NO:72; scrA gene SEQ ID NO:74; scrB gene SEQ ID NO:76, and scrR gene SEQ ID NO:78).

[0088] In a preferred embodiment, the microorganism according to the invention is a microorganism deposited on October 19, 2022 at the Collection Nationale de Cultures de Microorganismes, Pasteur Institute, 25 Rue du Docteur Roux, 75724 PARIS Cedex 15, FRANCE under the number CNCM I-5911.

[0089] A second object of the present invention relates to a method for the production of valine using the microorganism described herein, said method comprising the steps of: a) culturing a microorganism genetically modified for valine production as described herein in an appropriate culture medium containing a carbon source; b) recovering valine from the culture medium; Includes.

[0090] More particularly, the present invention relates to a method for improved valine fermentative production using the microorganisms described herein. According to the present invention, the terms "fermentation process", "fermentative production", "fermentation" or "cultivation" are used interchangeably to indicate the growth of a microorganism. This growth is generally carried out in a fermenter with an appropriate growth medium compatible with the microorganism used.

[0091] "Suitable culture medium" refers to a medium (e.g., a sterile liquid medium) that contains nutrients essential or beneficial for the maintenance and / or growth of cells, such as a carbon source or carbon substrate, a nitrogen source, such as peptone, yeast extract, meat extract, malt extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate; a phosphorus source, such as monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), such as magnesium salts, cobalt salts, and / or manganese salts; and growth factors, such as amino acids and vitamins. In particular, the mineral culture medium for E. coli may be of the same or similar composition as M9 medium (Anderson, 1946), M63 medium (Miller, 1992), or a medium as defined by Schaefer et al. (1999).

[0092] The term "source of carbon", "carbon source" or "carbon substrate" according to the present invention refers to any carbon source that can be metabolized by a microorganism, where the substrate contains at least one carbon atom. According to the present invention, the carbon source is preferably at least one carbohydrate, and optionally a mixture of at least two carbohydrates. CO2 is not a carbohydrate because it does not contain hydrogen.

[0093] The term "carbohydrate" refers to any carbon source that can be metabolized by a microorganism and contains at least one carbon atom, two hydrogen atoms, and one oxygen atom. The one or more carbohydrates can be selected from the group consisting of monosaccharides such as glucose, fructose, mannose, xylose, arabinose, galactose, disaccharides such as sucrose, cellobiose, maltose, lactose, oligosaccharides such as raffinose, stacchyose, maltodextrin, polysaccharides such as cellulose, hemicellulose, starch, methanol, formaldehyde, and glycerol. The preferred carbon source is arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose, or any polysaccharide such as starch, cellulose, or hemicellulose, or any combination thereof, more preferably glucose.

[0094] The term "recovery" as used herein refers to a step of separating or isolating the produced valine by using conventional laboratory techniques known to those skilled in the art. Recovering valine according to step b) of the method described herein may comprise the steps of filtration, desalting, cation exchange, liquid extraction, crystallization, or distillation, or combinations thereof. Valine may be recovered from both the culture medium and the microorganism, or only from either one of them. Preferably, valine is recovered at least from the culture medium. The amount of culture medium may be reduced, for example, by ceramic membrane filtration. Valine may further be recovered during the cultivation of the microorganism by in situ product recovery, including extractive fermentation, or after the fermentation has been terminated. The microorganism may be removed by passing through a device in which solid / liquid separation is performed, preferably a filter with a cut-off in the range of 5 to 200 kDa. It is also possible to use a centrifuge, a suitable sedimentation device, or a combination of these devices, and it is particularly preferred to first separate at least some of the microorganisms by sedimentation, and subsequently feed the fermentation broth from which the microorganisms have been at least partially removed, to an ultrafiltration device or a centrifugation device. After the microorganisms are removed, the valine present in the remaining culture medium can be recovered. The valine may be recovered separately from the microorganisms. Recovery of valine from the microorganisms may include, inter alia, dissolution or destruction by heating to induce the release of valine from the microorganisms.

[0095] In the second object of the present invention, the microorganism used according to the method for producing valine is the microorganism deposited on October 19, 2022 at the Collection Nationale de Cultures de Microorganismes, Pasteur Institute, 25 Rue du Docteur Roux, 75724 PARIS Cedex 15, FRANCE, under number CNCM I-5911.

[0096] More preferably, recovering valine according to step b) comprises at least a) clarification of the fermentation medium to remove insoluble organic impurities; b) a step of treatment of the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities; c) a step of evaporating water and crystallizing the product obtained; d) recovering valine; Includes.

[0097] The skilled artisan is able to define the culture conditions for the microorganism according to the invention. In particular, 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 and 37°C, even more preferably at about 37°C.

[0098] The process may be carried out as either a batch, fed-batch, or continuous process. It may be carried out under aerobic, microaerobic, or anaerobic conditions, or a combination thereof (e.g., aerobic followed by anaerobic conditions).

[0099] "Under aerobic conditions" means that oxygen is supplied to the culture by dissolving the gas in the liquid phase. This can be achieved by (1) sparging an oxygen-containing gas (e.g., air) into the liquid phase or (2) shaking the vessel containing the culture medium to transfer the oxygen contained in the headspace to the liquid phase. The main advantage of fermentation under aerobic conditions is that the presence of oxygen as an electron acceptor improves the ability of the strain to generate more energy in the form of ATP for intracellular processes. Thus, the strain has an improved overall metabolism.

[0100] Microaerobic conditions are defined as culture conditions with a low percentage of oxygen dissolved in the liquid phase (e.g., using a gas mixture containing 0.1-10% oxygen, with 100% nitrogen).

[0101] Anaerobic conditions are defined as culture conditions in which no oxygen is supplied to the culture medium. Strictly speaking, anaerobic conditions are obtained by bubbling an inert gas, such as nitrogen, through the culture medium to remove traces of other gases. Nitrate can be used as an electron acceptor to improve ATP production by the strain, improving metabolism.

[0102] The valine production by the microorganism in the culture broth can be determined unambiguously by standard analytical means known to those skilled in the art. As a non-limiting example, valine can be quantified using isocratic HPLC (Pleissner et al., 2011) or nuclear magnetic resonance methods. EXAMPLES

[0103] The present invention is further defined in the following examples. It should be understood that these examples, which show preferred embodiments of the present invention, are provided for illustrative purposes only. Those skilled in the art will easily understand that these examples are not limiting, and that various modifications, substitutions, omissions and changes can be made without departing from the scope of the present invention.

[0104] method The protocol used in the examples below is as follows. Protocol 1 (chromosomal modification by homologous recombination, selection of recombinants, and excision of the antibiotic cassette flanked by FRT sequences) as well as protocol 2 (transduction of phage P1) used in the present invention are fully described in patent application WO 2013 / 001055 (see in particular the section "Example Protocols" and Examples 1 to 8, which are incorporated herein by reference).

[0105] Protocol 3: Construction of recombinant plasmids. Recombinant DNA techniques are well described and known to those skilled in the art. Briefly, DNA fragments were PCR amplified using oligonucleotides (as may be defined by those skilled in the art), and genomic DNA of E. coli MG1655 or suitable synthetically synthesized fragments were used as a matrix. The DNA fragments and selected plasmids were digested with compatible restriction enzymes (as may be defined by those skilled in the art), then ligated and transformed into competent cells. The transformants were analyzed, and the desired recombinant plasmids were verified by DNA sequencing.

[0106] Protocol 4: Evaluation of L-valine fermentation performance The producing strain was evaluated in bioreactors using both media for valine production, MM_VAB10 and MM_VAB20 (Table 1), adjusted to pH 6.8. MM_VAB10 medium is dedicated to monitor the ability of the strain to produce valine at an early stage. MM_VBA20 medium is used to show the impact of gene optimization at high valine content. 50 mL preculture was cultured in rich medium (5 g.L -1 The bacteria were grown in LB medium (containing glucose) at 30°C for 16 hours, reaching an OD 600 The medium was used to inoculate the medium to a concentration of 0.5. When necessary, antibiotics were added to the medium (spectinomycin and chloramphenicol, each at a final concentration of 50 mg.L). -1 and 30 mg.L -1 ). The incubation temperature was 39 °C. The incubation was stopped when glucose was completely consumed within a maximum incubation time of 50 h. Extracellular amino acids were quantified by HPLC after OPA / Fmoc derivatization, and other related metabolites were analyzed using HPLC with refractive index detection (organic acids).

[0107] [Table 1]

[0108] In these cultures, the valine yield (YVal) was expressed as follows:

[0109]

number

[0110]

number

[0111] [Example 1] The valine-producing strain with more than one copy of ilvA gene had improved valine production performance. Construction of a valine-producing strain with alterable copy number of the ilvA gene Construction of strain 1 Following protocols 1, 2 and 3, E. coli MG1655 strain 1 was obtained sequentially. - knock out lactate dehydrogenase (IdhA gene, SEQ ID NO: 9), alcohol dehydrogenase (adhE gene, SEQ ID NO: 11) and methylglyoxal synthase (mgsA gene, SEQ ID NO: 13), - The acetohydroxyacid synthase I small regulatory subunit (ilvN gene, SEQ ID NO: 49) is converted to the valine feedback resistance (FBR) protein (IlvN * G20D, V21D and M22F substitutions in the FBR protein (SEQ ID NO: 52) - Park et al., 2012) and overexpression by replacing the native promoter with the artificial Ptrc promoter (Brosius et al., 1985); - on the pCL1920 vector (Lerner & Inouye, 1990), together with the cl857 allele (SEQ ID NO: 80) of the thermosensitive repressor of the lambda phage (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994), the following genes are overexpressed in an operon under the control of the PR promoter: - the vdh gene (SEQ ID NO: 53) encoding the valine dehydrogenase of Streptomyces aureofaciens (SEQ ID NO: 54, Uniprot A0A1E7N3I8); more precisely, the vdh gene was synthetically synthesized with codon usage optimized for E. coli (SEQ ID NO: 55), - the ilvD gene (SEQ ID NO: 45) encoding the IlvD dihydroxyacid dehydratase (SEQ ID NO: 46), - the ilvC gene (SEQ ID NO: 43) encoding the IlvC ketol-acid reductoisomerase (SEQ ID NO: 44), - The ilvBN genes, which code for both subunits of acetohydroxyacid synthase I, and the ilvN * FBR allele (ilvB gene SEQ ID NO: 47; ilvN * Gene SEQ ID NO:51), which gives rise to plasmid 1.

[0112] This strain has one copy of the ilvA gene (SEQ ID NO: 1) which encodes threonine dehydratase (SEQ ID NO: 2) which is endogenous.

[0113] Construction of strain 2 To construct strain 2, an additional copy of the ilvA gene and its promoter were integrated into a pseudogene of strain 1, preferentially selected from those cited in U.S. Patent Application Publication No. 2012 / 0252077, according to protocols 1 and 2.

[0114] Construction of strain 3 To construct strain 3, the ilvA gene and its promoter were cloned into the pACYC plasmid ( Bartolome et al., 1991 ) to generate plasmid 2, which was introduced into strain 1.

[0115] Improved valine production associated with copying the ilvA gene into strain 1 background Strains 1 to 3 were grown according to protocol 4. Valine productivity and yield were measured.

[0116] [Table 2]

[0117] In MM_VAB10 medium conditions, the valine productivity and valine yield of strain 1 with one copy of the ilvA gene are referred to as ≪criterion 1≫ and ≪criterion 2≫, respectively, and in MM_VAB20 medium conditions, the valine productivity and valine yield of strain 1 with one copy of the ilvA gene are referred to as ≪criterion 3≫ and ≪criterion 4≫, respectively. Compared to the appropriate criteria, the symbol ≪≒≫ indicates an increase of less than 10%, the symbol ≪++≫ indicates an increase of 30-100%, and the symbol ≪+++≫ indicates an increase of more than 100%.

[0118] As shown in Table 2, strain 2, which has two copies of the ilvA gene, has improved valine productivity regardless of the medium used compared to strain 1. Also, strain 3, which has more than two copies of the ilvA gene, has improved valine productivity and yield, especially in MM_VAB20 medium conditions.

[0119] These results are surprising since the ilvA gene is often deleted or attenuated in valine-producing strains described in the literature.

[0120] Improved valine production associated with copying the ilvA gene into the background of other valine-producing strains. The beneficial effect of ilvA copies in other genetic backgrounds has been demonstrated, more precisely in the following: - the W3110 E. coli strain described in Park et al., 2011 as "VAMF(pKBRilvBNmutCED, pTrc184ygaZHlrp)" in which the ilvA gene has been deleted, and - The W3110 E. coli strain, described as "VHY18" in Hao et al., 2020, which does not have any modifications to the ilvA gene, so this strain has one copy of the ilvA gene.

[0121] With the aim of increasing the copy number of the ilvA gene in both strains up to two, an additional copy of the ilvA gene and its promoter were added to both strains at the locus cited in US Patent Application Publication No. 2012 / 0252077 according to protocols 1 and 2, and the original ilvA gene was reconstituted at the endogenous locus in the Park strain.

[0122] Strains Park and Hao as well as the equivalent strain carrying two copies of the ilvA gene were cultivated as described under the respective conditions in Park et al., 2011 and Hao et al., 2020, and valine production was assessed as described in Protocol 4.

[0123] In the Park strain background, changing the ilvA gene from 0 to 2 copies slightly improves productivity, whereas in the Hao strain background, changing the ilvA gene from 1 to 2 copies improves productivity and yield by the same order of magnitude compared to strain 2 described in Example 1.

[0124] Furthermore, reconstitution of the ilvA gene in the Park strain is economically beneficial since it eliminates the need for the addition of leucine and isoleucine to the culture medium.

[0125] [Example 2] A point mutation in the argP gene improves valine production in a valine-producing strain carrying two copies of the ilvA gene Construction of strain 4 To construct strain 4, following protocols 1 and 2, the wild-type argP allele (SEQ ID NO: 3), which encodes a DNA-binding transcriptional dual regulator, was transformed into the argP mutant (SEQ ID NO: 6) encoding an ArgP mutant with an amino acid substitution of glutamic acid at position 128 to aspartic acid in strain 2. * It was replaced by the allele (SEQ ID NO:5).

[0126] argP * Improved valine production in strain 2 by alleles Strain 2 and strain 4 were grown according to protocol 4. Valine productivity and yield were measured.

[0127] [Table 3]

[0128] In MM_VAB10 medium conditions, the valine productivity and valine yield of strain 2 carrying the wild-type version of the argP gene are referred to as ≪criterion 5≫ and ≪criterion 6≫, respectively, and in MM_VAB20 medium conditions, the valine productivity and valine yield of strain 2 are referred to as ≪criterion 7≫ and ≪criterion 8≫, respectively. Compared to the appropriate criteria, the symbol ≪≒≫ indicates an increase of less than 10%, the symbol ≪++≫ indicates an increase of 30-100%, and the symbol ≪+++≫ indicates an increase of more than 100%.

[0129] As shown in Table 3, strain 4, which has a mutation in the argP gene (and two copies of the ilvA gene), has improved valine productivity, regardless of the medium used, compared to strain 2, which has a wild-type allele of the argP gene. The argP mutation does not affect the yield of valine.

[0130] [Example 3] A point mutation in the promoter of the fepA gene improves valine production in a valine-producing strain carrying two copies of the ilvA gene. Construction of strain 5 The gene fepA (SEQ ID NO: 82) encodes the FepA protein (SEQ ID NO: 83), an iron enterobactin outer membrane transporter. To construct strain 5, the wild-type fepA promoter sequence was replaced with the mutant one in strain 2, following protocols 1 and 2. The mutant promoter has a nucleobase substitution of T to C at position -3 from the transcription start (SEQ ID NO: 8).

[0131] Improved valine production in strain 2 by mutations in the fepA promoter Strain 2 and strain 5 were grown according to protocol 4. Valine productivity and yield were measured.

[0132] [Table 4]

[0133] In MM_VAB10 medium conditions, the valine productivity and valine yield of strain 2 carrying the wild-type version of the fepA promoter are referred to as ≪criterion 5≫ and ≪criterion 6≫, respectively, and in MM_VAB20 medium conditions, the valine productivity and valine yield of strain 2 are referred to as ≪criterion 7≫ and ≪criterion 8≫, respectively. The symbols ≪≒≫ indicate an increase of less than 10% and ≪++≫ indicates an increase of 30–100% compared to the appropriate criteria.

[0134] As shown in Table 4, strain 5, which has a mutation in the fepA promoter (and two copies of the ilvA gene), has improved valine productivity in MM_VAB20 medium conditions compared to strain 2, which has the wild-type sequence of the fepA promoter. The mutation in the fepA promoter does not affect the yield of valine.

[0135] [Example 4] A point mutation in the fepA promoter improves valine production in a valine-producing strain carrying two copies of the ilvA gene and a mutation in the argP gene. Construction of strain 6 To construct strain 6, the wild-type fepA promoter sequence was replaced with the sequence mutated in strain 4, following protocols 1 and 2. The mutated promoter has a T to C nucleobase substitution at position -3 from the transcription start (SEQ ID NO: 8).

[0136] Improved valine production in strain 4 by mutations in the fepA promoter Strain 4 and strain 6 were grown according to protocol 4. Valine productivity and yield were measured.

[0137] [Table 5]

[0138] In MM_VAB10 medium conditions, the valine productivity and valine yield of strain 4 carrying the mutated allele of argP and the wild-type sequence of the fepA promoter are referred to as ≪criterion 9≫ and ≪criterion 10≫, respectively, and in MM_VAB20 medium conditions, the valine productivity and valine yield of strain 4 are referred to as ≪criterion 11≫ and ≪criterion 12≫, respectively. The symbol ≪≒≫ indicates an increase of less than 10% and ≪+≫ indicates an increase of 10–30% compared to the appropriate criteria.

[0139] As shown in Table 5, strain 6, which has a mutation in the fepA promoter (and mutations in two copies of the ilvA and argP genes), has improved valine productivity in MM_VAB20 medium conditions compared to strain 4, which has the wild-type sequence of the fepA promoter. The fepA promoter mutation does not affect the yield of valine.

[0140] References Bantscheff et al., (2007), Analytical and Bioanalytical Chemistry, vol. 389(4): 1017-1031. Bartolome B, Jubete Y, Martinez E, de la Cruz Fet al., (1991), Gene., 1991 Jun 15;102(1):75-8. Burnette, (1981), Analytical Biochemistry, 112(2): 195-203. Carrier T & Keasling J., (1999), Biotechnol Prog., 15 (1): 58-64. Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Datsenko and Wanner, (2000), Proc Natl Acad Sci USA., 97: 6640-6645. Davis & Olsen., (2011), Mol. Biol. Evol., 28(1):211-221. Deml et al., (2011), J. Virol., 75(22): 10991-11001. Engvall and Perlman (1981), Immunochemistry, 8: 871-874. Fed-batch culture of Escherichia coli for L-valine production based on in silico flux response analysis. Graf et al., (2000), J. Virol., 74(22): 10 / 22-10826. Hao et al., (2020), Y, Ma Q, Liu X, Fan X, Men J, Wu H, Jiang S, Tian D, Xiong B, Xie X. Metab Eng., 2020 Nov; 62:198-206. High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation. Leuchtenberger, et al, (2005) Appl. Microbiol. Biotechnol. 69,1-8 Needleman and Wunsch (1970), J. Mol. Biol., 48(3), 443-453. Park et al., (2012), ACS synthetic biology, 1(11): 532-540 Park JH, Kim TY, Lee KH, Lee SYet al., (2011), Biotechnol Bioeng., 2011 Apr;108(4):934-46. Salis H., (2011), Methods Enzymol., 498:19-42 Segel I., (1993), Enzyme kinetics, John Wiley & Sons, pp. 44-54 and 100-112

Claims

1. A microorganism genetically modified for the production of valine, wherein the microorganism overexpresses the ilvA gene encoding threonine deaminase and / or exhibits increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in a corresponding wild-type microorganism, and further comprises a mutant argP gene encoding a DNA-binding transcriptional dual regulator.

2. 2. The microorganism according to claim 1, wherein the ilvA gene is overexpressed in the microorganism by overexpressing the ilvA gene from a plasmid, for example by modifying the promoter controlling the expression of the ilvA gene, by increasing the copy number of the ilvA gene present in the microorganism, or by increasing the amount of IlvA protein by improving the stability of ilvA mRNA or optimizing a ribosome binding site.

3. 3. The microorganism according to claim 1, wherein the ilvA gene is overexpressed in the microorganism by increasing the copy number of the ilvA gene present in the microorganism, preferably by providing at least two copies of the gene, more preferably by providing two copies of the gene.

4. 3. The microorganism according to claim 1 or 2, wherein the mutant argP gene has been modified to result in a substitution of the amino acid at position 128, preferably a substitution of Glu by Asp.

5. The microorganism described in claim 1 or 2, further comprising: an overexpression of the fepA gene encoding the iron enterobactin outer membrane transporter; and / or exhibiting increased iron enterobactin outer membrane transporter activity compared to the expression level and / or iron enterobactin outer membrane transporter activity in the corresponding wild-type microorganism.

6. 6. The microorganism according to claim 5, wherein the fepA gene is overexpressed by genetic modification, for example by modifying the promoter controlling the expression of the fepA gene, by increasing the copy number of the fepA gene present in the microorganism, or by overexpressing the fepA gene from a plasmid by improving the stability of fepA mRNA or by optimizing the ribosome binding site to increase the amount of FepA protein, preferably by mutating the promoter controlling the expression of the fepA gene.

7. 7. The microorganism of claim 6, wherein the fepA gene is overexpressed by at least one base substitution in a promoter sequence that controls expression of the fepA gene.

8. 3. The microorganism of claim 1 or 2, further comprising a deletion of at least one gene selected from the group consisting of IdhA, adhE, and mgsA.

9. vdh, ilvD, ilvC, ilvB, and ilvN ＊ 3. The microorganism of claim 1, further comprising overexpression of at least one gene selected from the group consisting of:

10. 3. The microorganism according to claim 1 or 2, wherein the microorganism belongs to the bacterial family Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcae or Lactobacillae, or to a fungus, such as a Hemiascomycetus, a filamentous fungus or a yeast.

11. The Enterobacteriaceae bacterium is Escherichia coli, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Bacillaceae family is Bacillus subtilis, the Streptococcusceae family is Streptococcus thermophiles, the Lactobacillaceae family is Lactobacillus lactis, and the hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica.

11. The microorganism of claim 10, wherein the filamentous fungus is Trichoderma rezeii or Aspergillus niger, and preferably the microorganism is Escherichia coli.

12. 2. The microorganism of claim 1, deposited on October 19, 2022 at the Collection Nationale de Cultures de Microorganismes, Pasteur Institute, 25 Rue du Docteur Roux, 75724 PARIS Cedex 15, FRANCE, under the number CNCM I-5911.

13. a) culturing a microorganism genetically modified for valine production according to any one of claims 1, 2 and 12 in an appropriate culture medium containing a carbon source; b) recovering valine from the culture medium; 1. A method for producing valine, comprising:

14. The step of recovering valine comprises at least a) clarification of the fermentation medium to remove insoluble organic impurities; b) treatment of the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities; c) evaporation of water and crystallization of the resulting product; d) recovering valine; 14. The method of claim 13, comprising: