Microorganisms and methods for improving valine production

Abstract

The present invention relates to a microorganism genetically modified for improved valine production wherein the microorganism overexpresses an ilvA gene encoding a threonine deaminase and / or exhibits increased threonine deaminase activity and comprises a mutant argP gene encoding a DNA binding transcription dual regulatory factor. The invention also relates to a method for producing valine using the microorganism.

Description

Technical Field

[0001] The present invention relates to a microorganism genetically modified for improved valine production, and to a method for improving valine production using the microorganism. Background Art

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

[0003] Branched-chain amino acids also act as precursors in the synthesis of herbicides and antibiotics such as polyketides. Unlike most other amino acids that are metabolized in the liver, branched-chain amino acids are primarily metabolized in the muscles, so they are used as an energy source for body movement.

[0004] Branched chain amino acids (BCAA) can be produced by chemical synthesis, extraction from protein hydrolysate or microbial fermentation. Among these technologies, fermentation is the most commonly used today due to the relevant economic and environmental advantages. In particular, fermentation provides a useful way to use abundant, renewable and / or cheap materials as the main carbon source. In addition, although both produce equimolar amounts of D- and L-enantiomers when using chemical synthesis, additional downstream separation of the L-enantiomer is required, fermentation only produces the L-enantiomer.

[0005] Among them, valine branched-chain amino acids are one of the nine essential amino acids that cannot be synthesized in mammals and are particularly important for the nutrition of humans and many livestock species. L-valine is usually produced by bacterial fermentation using Corynebacterium glutamicum and Escherichia coli.

[0006] To date, most BCAA production strains have been developed through random mutagenesis.

[0007] These microbial strains have the disadvantage that it is difficult to further improve the strains because it is difficult to understand their precise physiological metabolism. Therefore, in the art to which the present invention pertains, there is a need to develop microorganisms with high valine production capacity, thereby providing a novel method for producing valine at reduced cost. Summary of the invention

[0008] The present invention relates to a genetically modified microorganism for producing valine and a method for producing valine using the microorganism. The genetically modified microorganism for producing valine 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. In addition, the microorganism comprises a mutant argP gene encoding a DNA-binding transcriptional dual regulator.

[0009] Indeed, the inventors have surprisingly found that by overexpressing the ilvA gene and / or increasing the threonine deaminase activity, such microorganisms show improved valine production compared to prior art methods wherein the ilvA gene expression or the corresponding enzyme activity is not modified or is attenuated or even absent.

[0010] Preferably, in the genetically modified microorganism according to the present invention, the ilvA gene is overexpressed in the recombinant microorganism, such as by modifying the promoter regulating the expression of said ilvA gene, by increasing the copy number of said ilvA gene present in said microorganism, or by overexpressing said ilvA gene from a plasmid, by increasing the stability of ilvA mRNA or by optimizing the ribosome binding site to increase the amount of IlvA protein, preferably by mutating the promoter regulating the expression of said ilvA gene.

[0011] 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.

[0012] Preferably, the microorganism further comprises further overexpression of a fepA gene encoding a ferric ferrous enterobactin outer membrane transporter and / or exhibits increased ferric ferrous enterobactin outer membrane transporter activity compared to the expression level and / or ferric ferrous enterobactin outer membrane transporter activity in a corresponding wild-type microorganism.

[0013] Preferably, the microorganism further comprises a deletion of at least one gene selected from the group consisting of ldhA, adhE and mgsA.

[0014] Preferably, the microorganism further comprises overexpression of at least one gene selected from the group consisting of vdh, ilvD, ilvC, ilvB and ilvN*.

[0015] Preferably, the microorganism belongs to the bacteria Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcae or Lactobacillae, or to the fungal family, such as Hemiascomycetus, filamentous fungi or yeast.

[0016] Preferably, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacterium family bacterium is Corynebacterium glutamicum, or the Bacillaceae family is Bacillus subtilis, the Streptococcus family is Streptococcus thermophiles, the Lactobacillaceae family is Lactobacillus lactis, the Hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, and the filamentous fungus is Trichoderma rezeii or Aspergillus niger, more preferably wherein the microorganism is Escherichia coli.

[0017] Preferably, the microorganism is a microorganism numbered CNCM I-5911, deposited on October 19, 2022 at the National Collection of Microorganisms of the Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

[0018] The present invention further includes a method for producing valine, the method comprising the steps of:

[0019] a) culturing a microorganism genetically modified to produce valine as described in any one of the embodiments provided herein in a suitable culture medium comprising a carbon source, and

[0020] b) recovering valine from the culture medium.

[0021] Preferably, the recovery of valine comprises at least the following steps:

[0022] a) clarifying the fermentation medium to remove insoluble organic impurities,

[0023] b) treating the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities,

[0024] c) evaporating the water and crystallizing the product obtained,

[0025] d) recovering valine.

[0026] 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

[0027] Before describing the present invention in detail, it should be understood that the present invention is not limited to the microorganism and / or method specifically exemplified, and can certainly change.It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments of the present invention, and are not intended to be restrictive.The present invention will only be limited by the appended claims.

[0028] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety, whether above or below. In addition, unless otherwise indicated, the practice of the present invention employs conventional microbiology and molecular biology techniques within the skill of the art. Such techniques are well known to those skilled in the art and are fully explained in the literature.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the 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.

[0030] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a microorganism" includes a plurality of such microorganisms, and reference to "an endogenous gene" is a reference to one or more endogenous genes, and so forth.

[0031] The terms “comprise”, “comprises” and “comprising” are used herein in an inclusive sense, ie to specify the presence of stated features in various embodiments of the invention but do not preclude the presence or addition of further features.

[0032] A first aspect of the present invention relates to a microorganism genetically modified for producing valine.

[0033] As used herein, the term "microorganism" refers to a living microscopic organism, which can be a unicellular or multicellular organism and can generally be found in nature. In the context of the present invention, the microorganism is preferably a bacterium, a yeast or a fungus. Preferably, the microorganism of the present invention is selected from the family Enterobacteriaceae, Corynebacterium, Bacillaceae, Streptococcus or Lactobacillaceae, or is selected from the family Fungi (such as Hemiascomycetes, filamentous fungi or yeast). More preferably, the microorganism of the present invention is a species of Escherichia, Corynebacterium, Bacillus, Streptococcus, Lactobacillus. Even more preferably, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacterium family bacterium is Corynebacterium glutamicum, or the Bacillaceae family is Bacillus subtilis, the Streptococcus family is Streptococcus thermophilus, the Lactobacillaceae family is Lactobacillus lactis, the Hemiascomycetes 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.

[0034] The terms "recombinant microorganism" or "genetically modified microorganism" are used interchangeably herein and refer to a microorganism or microorganism strain that has been genetically modified or genetically engineered. This means that, according to the ordinary meaning of these terms, the microorganism of the present invention does not exist in nature and is genetically modified when compared to the "parent" microorganism from which it is derived. The "parent" microorganism may exist in nature (i.e., a wild-type microorganism) or may have been previously modified. The recombinant microorganism of the present invention can be modified, in particular, by the introduction, deletion and / or modification of genetic elements. Such modifications can be carried out, for example, by the following means: for example, genetic engineering; adaptation, wherein the microorganism is cultivated under conditions where a specific stress is imposed on the microorganism and mutagenesis is induced; and / or by combining directed mutagenesis and evolution under a specific selection pressure to force the development and evolution of metabolic pathways.

[0035] Microorganisms can be modified to regulate the expression level of endogenous genes or the activity of corresponding enzymes or transcription factors in particular. The term "endogenous gene" means that the gene is present in the microorganism before any genetic modification. Endogenous genes can be overexpressed by introducing heterologous sequences to supplement or replace endogenous regulatory elements. It is also possible to increase or weaken the activity of endogenous gene expression levels, protein expression levels or encoded proteins by introducing mutations into the coding sequence of the gene or into non-coding sequences. When no modification is made in the corresponding amino acids, these mutations can be synonymous, or when the corresponding amino acids are changed, these mutations can be non-synonymous. Synonymous mutations have no effect on the function of the translated protein, but if the mutant sequence is located in the binding site of the regulatory factor, the regulation of the corresponding gene or even other genes may be affected. Non-synonymous mutations may affect the function or activity and regulation of the translated protein, depending on the nature of the mutant sequence.

[0036] In particular, the mutation in the non-coding sequence can be located upstream of the coding sequence (that is, in the promoter region, in enhancer, silencer or insulator region, in a specific transcription factor binding site) or downstream of the coding sequence. The mutation introduced in the promoter region can be in a core promoter, a proximal promoter or a distal promoter. The mutation can be by using, for example, the site-directed mutagenesis of polymerase chain reaction (PCR), by, for example, random mutagenesis techniques via mutagen (ultraviolet light or chemical agent, such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS)), DNA reorganization, fallible PCR or use the culture conditions that apply specific stress to microorganisms and induce mutagenesis to introduce. In the region located upstream of the gene, inserting one or more supplementary nucleotides can especially regulate gene expression.

[0037] A specific way to regulate the expression of endogenous genes is to exchange the endogenous promoter of the gene (e.g., wild-type promoter) 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., derived from the same species) or exogenous (i.e., derived from a different species). It is entirely within the capabilities of those skilled in the art to select an appropriate promoter to regulate the expression of an endogenous gene. Such a promoter can, for example, be a Ptrc, Ptac or Plac promoter, or a PR or PLλ promoter. The promoter can be "induced" by a specific compound or by a specific external condition (such as temperature or light).

[0038] A specific way to modulate the activity of an endogenous protein is to introduce a non-synonymous mutation into the coding sequence of the corresponding gene, for example according to any of the methods described above. Non-synonymous amino acid mutations present in a transcription factor can significantly change the binding affinity of the transcription factor to a cis-element, alter the binding of a ligand to the transcription factor, etc.

[0039] Microorganism can also be genetically modified to express one or more foreign (i.e., heterologous) genes, so as to express or overexpress corresponding gene products (e.g., enzymes). As used herein, "foreign" or "heterologous" genes refer to genes encoding proteins or polypeptides introduced into microorganisms, wherein the genes are not naturally present in the microorganisms. As used herein, "heterologous genes" also refer to genes that are endogenous to microorganisms (i.e., present in microorganisms before any genetic modification) but are not introduced at the position where the endogenous gene is located when introduced into the microorganism. More particularly, in the case where the expression of the endogenous gene itself is reduced compared to the microorganisms naturally presenting the gene in the microorganism (e.g., due to mutation, complete or partial deletion of the gene, modification of gene transcription regulation, etc.), the heterologous gene can be an endogenous gene. In particular, the endogenous gene may no longer be expressed or may be expressed at very low levels. Foreign genes can be directly integrated into the chromosome of a microorganism, or are expressed extrachromosomally in a microorganism by plasmids or vectors. For successful expression, one or more foreign genes must be introduced into the microorganism together with all regulatory elements necessary for their expression, or one or more foreign genes must be introduced into the microorganism that already contains all regulatory elements necessary for their expression. Genetic modification or transformation of microorganisms with one or more foreign genes is a routine task for those skilled in the art.

[0040] One or more copies of a given foreign gene can be introduced into the chromosome by methods well known in the art (such as by genetic recombination). When a gene is expressed outside the chromosome, it can be carried by a plasmid or a vector. Different types of plasmids are especially available, and they may be different in replication orgin and / or in the copy number in the cell. For example, the microorganism transformed by the plasmid can contain 1 to 5 copies, about 20 copies or even up to 500 copies of the plasmid, depending on the property of the selected plasmid. A variety of plasmids with different replication orgins and / or copy numbers are well known in the art and can be easily selected for such purposes by skilled personnel, including for example pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2 or pPLc236.

[0041] It should be understood that in the context of the present invention, when an exogenous gene encoding a protein of interest is expressed in a microorganism, a synthetic form of the gene can preferably be constructed by replacing non-preferred codons or less preferred codons with preferred codons of the microorganism encoding the same amino acid. In fact, it is well known in the art that codon usage differs between microbial species, and this may affect the level of recombinant expression of the protein of interest. In order to overcome this problem, codon optimization methods have been developed and are widely described by Graf et al. (2000), Deml et al. (2001) and Davis and Olsen (2011). Several software programs have been developed in particular for codon optimization determination, such as Software (Lifetechnologies) or OptimumGene TM Software (GenScript). In other words, the foreign gene encoding the target protein is preferably codon-optimized for expression in a microorganism.

[0042] The term "expression", "overexpressing" or "overexpression" of a target protein (such as an enzyme) refers herein to an increase in the expression level and / or activity of the protein in a microorganism compared to a corresponding parent microorganism (also referred to as a corresponding wild-type microorganism) that does not contain the modification present in the genetically modified microorganism. In some cases, the expression level can be similar to the expression level of the parent microorganism. In other cases, the expression level can be superior to the expression level of the parent microorganism. In the case where the parent microorganism does not contain the target protein, the term "expression" or "overexpression" refers to the presence of the target protein compared to the absence of the target protein in the parent microorganism.

[0043] In contrast, the term "attenuating" or "attenuation" of a protein of interest refers to a reduction in the expression level and / or activity of the protein in a microorganism compared to the parent microorganism. The attenuation of expression may be particularly due to the replacement of a wild-type promoter with a weaker natural or synthetic promoter, or the use of an agent that reduces gene expression, such as antisense RNA or interfering RNA (RNAi), and more particularly small interfering RNA (siRNA) or short hairpin RNA (shRNA). Promoter exchange may be achieved in particular by homologous recombination techniques (Datsenko and Wanner, 2000). Complete attenuation of the expression level and / or activity of a protein of interest means that expression and / or activity are eliminated, and therefore, the expression level of the protein is zero. Complete attenuation of the expression level and / or activity of a protein of interest may be due to complete inhibition of gene expression. This inhibition may be inhibition of gene expression, deletion of all or part of a promoter region necessary for gene expression, or deletion of all or part of a gene coding region. The deleted gene may be replaced in particular by a selectable marker gene, which is conducive to the identification, separation and purification of the modified microorganism. As a non-limiting example, inhibition of gene expression can be achieved by homologous recombination technology, which is well known to those skilled in the art (Datsenko and Wanner, 2000).

[0044] Thus, modulation of the expression level of one or more proteins can be performed by altering the expression of one or more endogenous genes encoding said proteins within the microorganism as described above or by introducing one or more heterologous genes encoding said proteins into the microorganism.

[0045] As used herein, the term "expression level" refers to the amount (e.g., relative amount, concentration) of the target protein (or gene encoding the protein) expressed in a microorganism, which can be measured by methods well known in the art. Gene expression levels can be measured by various known methods including Northern blotting, quantitative RT-PCR, etc. Alternatively, the expression level of the protein encoded by the gene can be measured, for example, by SDS-PAGE, HPLC, LC / MS and other quantitative proteomics techniques (Bantscheff et al., 2007), or when antibodies to the protein are available, by Western blotting-immunoblotting (Burnette, 1981), enzyme-linked immunosorbent assay (e.g., ELISA) (Engvall and Perlman, 1971), protein immunoprecipitation, immunoelectrophoresis, etc. The copy number of the expressed gene can be, for example, by limiting chromosomal DNA, and then using probes based on gene sequences to perform Southern blotting, fluorescent in situ hybridization (FISH), RT-qPCR, etc. to quantify.

[0046] Overexpression of a given gene or corresponding protein can be verified by comparing the expression level of said gene or protein in the genetically modified organism with the expression level of the same gene or protein in a control microorganism without genetic modification (ie, the parental microorganism).

[0047] The term "activity" or "function" as used in the context of an enzyme refers to the reaction catalyzed by the enzyme for converting its corresponding substrate or substrates into another molecule or molecules (i.e., one or more products). 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, incorporated herein by reference, particularly at pages 44-54 and 100-112.

[0048] The microorganisms provided herein that are genetically modified for improving the production of valine overexpress the ilvA gene encoding threonine deaminase and / or exhibit increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism. In fact, the inventors have unexpectedly shown that the above genetic modifications improve the production of valine, especially in terms of productivity and yield, compared to the parent microorganism that does not comprise these modifications. The improved valine production in this microorganism is unexpected because the ilvA gene expression or threonine deaminase activity is usually not modified or weakened and even missing, i.e., valine production is not improved in the prior art methods.

[0049] In the microorganisms provided herein that are genetically modified for producing valine, any well-known prior art methods can be used to overexpress ilvA gene expression or increase threonine deaminase activity. "Overexpression" or "overexpressing" are also used to indicate an increase in gene transcription in a microorganism. Increasing the transcription of a gene can be achieved by increasing the copy number of the gene and / or using a promoter that causes a higher level of expression of the gene.

[0050] For example, the ilvA gene can be overexpressed by modifying the promoter regulating the expression of the ilvA gene, increasing the copy number of the ilvA gene present in the microorganism, or overexpressing the ilvA gene from a plasmid, improving the stability of ilvA mRNA or increasing the amount of IlvA protein by optimizing the ribosome binding site.

[0051] Preferably, the ilvA gene expression is overexpression, and in particular by increasing the copy number of the ilvA gene present in the microorganism.

[0052] In order to increase the copy number of a gene in a microorganism, the gene is chromosomally encoded or extrachromosomally encoded. When the gene is located on the chromosome, several gene copies can be introduced into the chromosome by recombination methods known to experts in the field (including gene replacement). When the gene is located extrachromosomally, it can be carried by different types of plasmids, which have different origins of replication, so their copy numbers in the cell are different, as described above.

[0053] 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 15 copies can also be considered. More preferably, from 2 to 5 copies of the ilvA gene are present in the genetically modified microorganism of the present invention. Most preferably, the ilvA gene is overexpressed in the genetically modified microorganism, resulting in the production of two copies of the gene.

[0054] Increasing the translation of mRNA can be achieved by modifying the ribosome binding site (RBS). RBS is a sequence on mRNA that is bound by the ribosome when protein translation is initiated. It can be the 5' cap of mRNA in eukaryotes, the region 6-7 nucleotides upstream of the start codon AUG in prokaryotes (called the SD sequence), or the internal ribosome entry site (IRES) in viruses. By modifying this sequence, the protein translation initiation rate can be changed, its production rate can be changed proportionally, and its activity in the cell can be controlled. Depending on the nature of the mRNA, the same RBS sequence will not have the same impact. The strength of the RBS sequence can be optimized to achieve the target translation initiation rate by using the software RBS calculator (Salis, 2011).

[0055] Improving mRNA stability can be achieved by reducing mRNA turnover, which can 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).

[0056] In a preferred embodiment, the microorganism overexpresses the ilvA gene encoding the threonine deaminase of SEQ ID NO: 2 or a functional fragment or functional variant thereof.

[0057] The term "functional fragment" of a reference protein having a biological activity of interest (e.g., an enzyme having threonine deaminase activity), as used herein, refers to a portion of the amino acid sequence of an enzyme that contains at least all regions necessary to exhibit the biological activity of the protein. These portions of the sequence can have various lengths, provided that the biological activity of the reference amino acid sequence is retained by the portion. In other words, the functional fragments of the enzymes provided herein have enzymatic activity.

[0058] "Functional variants" of the enzymes described herein (e.g., enzymes having threonine deaminase activity) include, but are not limited to, enzymes having an amino acid sequence that is at least 60% identical to the amino acid sequence encoding the corresponding reference enzyme after alignment. 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, the 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. As a non-limiting example, the means for determining sequence identity are further provided below.

[0059] Increased enzyme activity can also be obtained by increasing protein catalytic efficiency or decreasing protein turnover or decreasing messenger RNA (mRNA) turnover or increasing gene transcription or increasing mRNA translation.

[0060] Improving protein catalytic efficiency means increasing kcat and / or reducing Km for a given substrate and / or a given cofactor, and / or increasing Ki for a given inhibitor. kcat, Km and Ki are Michaelis constants that can be determined by a person skilled in the art (Segel, 1993). Reducing protein turnover means stabilizing the protein. Methods for improving protein catalytic efficiency and / or reducing protein turnover are well known to those skilled in the art. These methods include rational engineering by sequence and / or structural analysis and directed mutagenesis, as well as random mutagenesis and screening. Mutations can be introduced by site-directed mutagenesis by commonly used methods such as polymerase chain reaction (PCR), or by random mutagenesis techniques such as the use of mutagens (ultraviolet light, or chemical reagents such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), or using PCR techniques (DNA shuffling or error-prone PCR). Stabilizing proteins can also be achieved by adding a peptide sequence called a "tag" to 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.

[0061] Unless otherwise specified, otherwise in this article, use the title of corresponding gene in Escherichia coli to identify gene and protein (for example, the Escherichia coli K12 MG1655 with Genbank accession number U00096.3). However, in some cases, the use of these titles has more general meaning according to the present invention and contains all corresponding genes and proteins in microorganisms. For non-endogenous (i.e. heterologous) gene and protein such as IlvA for microorganism of the present invention as described herein, the situation is especially so. As a specific example, and as indicated above, this paper includes the functional variant of IlvA, and its mutant and functional fragment. Specific aspects are described in further detail below.

[0062] PFAM (Protein Families Database of Alignments and Hidden Markov Models; http: / / www.sanger.ac.uk / Softw are / Pfam / ) represents a large collection of protein sequence alignments. Each PFAM can visualize multiple alignments, view protein domains, evaluate distribution between organisms, gain access to other databases, and visualize known protein structures.

[0063] COGs (Clusters of Orthologous Proteins; http: / / www.ncbi.nlm.nih.gov / COG / ) were obtained by comparing protein sequences from 43 completely sequenced genomes representing 30 major phylogenetic lineages. Each COG was defined by at least three lineages, which allowed the identification of previously conserved domains.

[0064] Means for identifying similar sequences and their percent identities are well known to those skilled in the art and include in particular the BLAST program, which can be used from the website http: / / www.ncbi.nlm.nih.gov / BLAST / with the default parameters indicated on the website. The sequences obtained can then be developed (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 these websites.

[0065] Using the references given on GenBank about known genes, those skilled in the art can determine equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously carried out using consensus sequences, which can be determined by comparing sequences with genes derived from other microorganisms and designing degenerate probes to clone the corresponding gene in another organism. These conventional methods of molecular biology are well known to those skilled in the art.

[0066] Sequence identity between amino acid sequences can be determined by comparing positions in each of the sequences, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base, then the sequences are identical at that position. The degree of sequence identity between proteins varies with the number of identical amino acid residues at positions shared by the sequences of the proteins.

[0067] As a non-limiting example, in order to determine the percent identity between two amino acid sequences, the sequences are aligned for optimal comparison. For example, a room can be introduced into the sequence of a first amino acid sequence for optimal comparison with a 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 the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position.

[0068] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Thus, % identity = number of identical positions / total number of overlapping positions x 100.

[0069] Optimal alignment of sequences can be performed by the global alignment algorithm of Needleman and Wunsch (1972), by a computerized implementation of the algorithm (such as CLUSTAL W), or by visual inspection. The best alignment produced by each method (i.e., resulting in the highest percent identity between the compared sequences) is selected.

[0070] In other words, the percent sequence identity is calculated by comparing the two optimally aligned sequences, determining the number of positions where the identical amino acid occurs 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 percent sequence identity.

[0071] The above definitions and preferred embodiments relating to functional fragments and functional variants of proteins apply mutatis mutandis to nucleotide sequences, such as genes encoding the protein of interest (ie, an enzyme having threonine deaminase activity).

[0072] In addition to the above modifications, the genetically modified microorganism of the present invention can also include one or more other modifications in the following ones. The modifications are advantageous because they can significantly further improve the production, titer and / or productivity of valine. One or more of the modifications can significantly promote the synthesis of valine, suppress valine from being used as a substrate in downstream metabolic pathways, promote the stable accumulation of valine, or suppress the toxic accumulation of valine in the microorganism.

[0073] According to the present invention, the microorganism further comprises a mutated argP gene encoding a DNA-binding transcription dual regulator.

[0074] 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 a corresponding position. The most preferred embodiment in this regard is that the Glu at position 128 or a corresponding position is replaced by Asp (also referred to herein as "argP * Mutant"). The positions of the indicated amino acid residues correspond to the positions provided in SEQ ID NO:4. In addition, the argP gene encodes a transcriptional regulator that causes various regulation of target gene expression (from overexpression to reduced expression) by directly binding to the promoter or control region of the target gene. Among the target genes, the following genes can be mentioned: gdhA, dapB, dapD, lysP, lysA, lysC, asd, dnaAN-recF, nrdAB-yfaE, argP, argO, argK... Therefore, compared with the usual regulation in the case where argP is not mutated (i.e. wild type), all modifications to the expression of the target gene due to the substitution of the amino acid at position 128 or the corresponding position of argP should be considered to have the same improving effect on valine production and yield as compared with the substitution of the same position of argP itself.

[0075] Corresponding positions can be clearly determined by those skilled in the art using manual alignment or by using an alignment program (e.g., BLASTP). Corresponding positions can also be based on structural alignments, such as by using a computer simulation alignment of a protein structure. The fact that the amino acids of a polypeptide correspond to the amino acids in the disclosed sequence refers to the fact that when comparing the polypeptide and the disclosed sequence, a standard alignment calculation method (such as the GAP calculation method) is used. When conservative amino acids are aligned so that the sequence has the greatest identity or homology, the corresponding amino acids can be clearly identified. As used herein, "at a corresponding position" refers to the target position (i.e., nucleotide base or amino acid residue numbering) in a nucleic acid molecule or protein relative to a position in a reference nucleic acid molecule or protein. The target position 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 in other species, etc. The corresponding position can be determined by comparing and aligning the sequence so that the number of paired nucleotides or amino acid residues is maximized. For example, the identity between the sequences can be greater than 95%, 96%, 97%, 98%, or more particularly greater than 99%. The target position is then given the numbering assigned in the sequence of the reference nucleic acid molecule or polypeptide.

[0076] According to the invention, therefore, the microorganism overexpresses the ilvA gene encoding a threonine deaminase and / or exhibits an increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism, together with a mutated argP gene encoding a DNA-binding transcriptional dual regulator as described above.

[0077] According to another preferred embodiment, the microorganism further 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.

[0078] As mentioned herein, any method known in the art can be used for overexpressing the fepA gene in the microorganism according to the present invention. Therefore, any technical means that lead to increased fepA gene expression or increased ferric iron enterobactin outer membrane transporter activity compared with the expression level and / or ferric iron enterobactin outer membrane transporter activity in the corresponding wild-type microorganism can be used to genetically modify the microorganism in the context of the present invention. In particular and only as an example, by genetic modification, such as by modifying the promoter that regulates 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 mutation regulating the promoter of the expression of the fepA gene. In fact, any modification that can lead to increased fepA gene expression or increased ferric iron enterobactin outer membrane transporter activity compared with the expression level and / or ferric iron enterobactin outer membrane transporter activity in the corresponding wild-type microorganism is included in the scope of the present invention.

[0079] Preferably, the microorganism further comprises increasing the expression of the fepA gene by performing at least one base substitution in the promoter sequence regulating the expression of the fepA gene. More preferably, the at least one base substitution, and in particular one base substitution, is performed immediately downstream of the "-10 box" (also referred to as the "Pribnow box"), which is located about 10 nucleotide pairs upstream of the transcription start point and consists of 6 nucleotides, optionally with some variants, but generally with the sequence TATAAT. In particular, a nucleotide substitution is performed 3 nucleotides upstream of the transcription start of the promoter controlling the expression of the fepA gene, as shown in SEQ ID NO:7. More preferably, the thymine nucleotide at position -3 from the transcription start of the fepA promoter is replaced by a cytosine nucleotide, as shown in SEQ ID NO:8.

[0080] In another advantageous embodiment of the present invention, the microorganism overexpresses the ilvA gene encoding a threonine deaminase and / or exhibits an increased threonine deaminase activity compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism, together with overexpression of the fepA gene encoding a ferric iron enterobactin outer membrane transporter and / or increased ferric iron enterobactin outer membrane transporter activity as described above.

[0081] In a still advantageous embodiment of the present invention, the microorganism overexpresses the ilvA gene encoding threonine deaminase and / or exhibits increased threonine deaminase activity, together with a mutant argP gene encoding a DNA-binding transcriptional dual regulator as described above, together with overexpression of the fepA gene encoding a ferric iron enterobactin outer membrane transporter as described above and / or increased ferric iron enterobactin outer membrane transporter activity, compared to the expression level and / or threonine deaminase activity in the corresponding wild-type microorganism.

[0082] According to still another preferred embodiment, the microorganism further comprises attenuation of the 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 phosphotransacetylase (Pta) and / or branched-chain amino acid transporter (BrnQ and LivKHMGF). The genes are especially endogenous in Escherichia coli.

[0083] Preferably, LdhA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 10. Preferably, AdhE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 12. Preferably, MgsA has at least 80%, 90%, 95% or 100% sequence similarity or sequence 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 sequence 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 sequence 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 sequence identity with the sequence of SEQ ID NO: 28. Preferably, Pta has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 30. Preferably, BrnQ has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with 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 sequence identity with the sequences of SEQ ID NO: 34, 36, 38, 40 and 42, respectively.

[0084] Preferably, the reduction in expression is caused by the partial or complete deletion of genes encoding the protein (i.e., ldhA, adhE, mgsA, frdABCD, pflAB, ackA-pta, brnQ and / or livKHMGF genes). Preferably, the genetically modified microorganism of the present invention further comprises at least one deletion selected from the group consisting of ldhA, adhE and mgsA.

[0085] Preferably, the ldhA 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 gene has 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.

[0086] The microorganism for producing valine may further comprise overexpression of one or more of 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.

[0087] 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 NO:48 and 52, respectively, wherein when the sequence is not 100% identical to SEQ ID NO:52, IlvN* comprises the substitutions G20D, V21D and M22F. Preferably, Vdh has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:54. Preferably, IlvE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to the sequence of SEQ ID NO: 57. Preferably, YgaZ and YgaH have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to the sequences of SEQ ID NO: 59 and 61, respectively.

[0088] Preferably, the overexpression of the one or more proteins is caused by overexpression of genes encoding said proteins, i.e. ilvC, ilvD and / or ilvBN* genes. Preferably, the genetically modified microorganism of the present invention further comprises overexpression of at least one gene selected from the group consisting of vdh, ilvD, ilvC, ilvB and ilvN*.

[0089] 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 have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO:47 and 51, respectively, wherein the amino acids encoded by the ilvN* gene have substitutions G20D, V21D and M22F relative to the wild-type protein having the sequence 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 to the sequences of SEQ ID NOs: 58 and 60, respectively.

[0090] As mentioned above, the microorganism of the present invention may belong to the bacterial, fungal or yeast family.

[0091] Preferably, the microorganism belongs to the bacterial family Enterobacteriaceae, Corynebacteraceae, Bacillaceae, Streptococcus or Lactobacillaceae, or to the fungal family, such as Hemiascomycetes, filamentous fungi or yeast.

[0092] According to a preferred embodiment, the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacteraceae bacterium is Corynebacterium glutamicum, or the Bacillaceae is Bacillus subtilis, the Streptococcus family is Streptococcus thermophilus, the Lactobacillaceae is Lactobacillus lactis, the Hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, and the filamentous fungus is Trichoderma reesei or Aspergillus niger, and more preferably the microorganism is Escherichia coli.

[0093] Furthermore, the microorganism according to the present invention may be genetically modified to comprise a modified endogenous gene / enzyme or a heterologous gene / enzyme. Preferably, the microorganism comprises an endogenous gene or enzyme having threonine deaminase activity, more preferably an endogenous ilvA gene encoding a threonine deaminase.

[0094] In a further aspect, when the microorganism as described herein is unable to use sucrose as a carbon source, the microorganism is modified to be able to use sucrose as a carbon source. Preferably, proteins involved in sucrose import and metabolism are overexpressed. Preferably, the following proteins are overexpressed:

[0095] - CscB sucrose permease (SEQ ID NO: 63), CscA sucrose hydrolase (SEQ ID NO: 65), CscK fructokinase (SEQ ID NO: 67) and CscRcsc-specific repressor (SEQ ID NO: 69), or

[0096] - The ScrA II enzyme of the phosphoenolpyruvate-dependent phosphotransferase system (SEQ ID NO:75), the ScrK gene encoding the ATP-dependent fructokinase (SEQ ID NO:71), the ScrB sucrose 6-phosphohydrolase (invertase) (SEQ ID NO:77), the ScrY sucrose porin (SEQ ID NO:73), and the ScrR sucrose operon repressor (SEQ ID NO:79).

[0097] Preferably, a gene encoding the protein is overexpressed according to one of the methods provided herein. Preferably, the E. coli microorganism overexpresses:

[0098] - heterologous cscBKAR genes of Escherichia 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

[0099] - Heterologous scrKYABR genes of Salmonella sp (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).

[0100] In a preferred aspect, the microorganism according to the invention is a microorganism numbered CNCM I-5911, deposited on October 19, 2022 at the National Collection of Microorganisms of the Pasteur Institute, 25 Rue du Docteur Roux, Cedex 15, 75724 Paris, France.

[0101] A second object of the present invention relates to a method for producing valine using the microorganisms described herein. The method comprises the following steps:

[0102] a) cultivating a microorganism genetically modified for producing valine as described herein in a suitable medium comprising a carbon source, and

[0103] b) recovering valine from the culture medium.

[0104] More particularly, the present invention relates to methods for improving the fermentative production of valine using the microorganisms described herein. According to the present invention, the terms "fermentation process", "fermentative production", "fermentation" or "cultivation" are used interchangeably to refer to the growth of microorganisms. Such growth is typically carried out in a fermentor with an appropriate growth medium suitable for the microorganism being used.

[0105] "Suitable medium" refers to a medium (e.g., a sterile liquid medium) containing nutrients necessary or beneficial to 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 potassium dihydrogen phosphate or dipotassium hydrogen 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 inorganic medium for Escherichia coli can have the same or similar components as M9 medium (Anderson, 1946), M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al. (1999).

[0106] According to the present invention, the term "carbon source", "carbon source" or "carbon substrate" refers to any carbon source that can be metabolized by a microorganism, wherein the substrate contains at least one carbon atom. According to the present invention, the carbon source is preferably at least one carbohydrate, and in some cases a mixture of at least two carbohydrates. CO2 is not a carbohydrate because it does not contain hydrogen.

[0107] 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. One or more carbohydrates can be selected from: monosaccharides such as glucose, fructose, mannose, xylose, arabinose, galactose, etc.; disaccharides such as sucrose, cellobiose, maltose, lactose, etc.; oligosaccharides such as raffinose, stachyose, maltodextrin, etc.; polysaccharides such as cellulose, hemicellulose, starch, etc.; methanol, formaldehyde and glycerol. Preferred carbon sources are arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose or any polysaccharide (such as starch, cellulose or hemicellulose) or any combination thereof, more preferably glucose.

[0108] The term "recovery" as used herein refers to the process of separating or isolating the produced valine using conventional laboratory techniques known to those skilled in the art. Recovering valine according to step b) of the method described herein may include steps of filtration, desalting, cation exchange, liquid extraction, crystallization or distillation or a combination thereof. Valine can be recovered from both the culture medium and the microorganism, or only from one or the other. Preferably, valine is recovered at least from the culture medium. For example, the volume of the culture medium can be reduced by filtration through a ceramic membrane. Valine can also be recovered during the culture of the microorganism by in situ product recovery including extractive fermentation, or after the fermentation is completed. The microorganism can be removed in particular by passing through a device, preferably through a filter with a cut-off in the range of 5 to 200 kDa, where solid / liquid separation occurs. It is also feasible to use a centrifuge, a suitable sedimentation device or a combination of these devices, particularly preferably first separating at least part of the microorganism by sedimentation, and then feeding the fermentation broth from which the microorganism has been at least partially removed to an ultrafiltration or to a centrifugal device. After the microorganism has been removed, the valine present in the remaining culture medium can be recovered. Valine can be recovered from the microorganism alone. Recovery of valine from microorganisms may involve, inter alia, cleavage or disruption by heating to induce release of valine from the microorganism.

[0109] In the second object of the present invention, the microorganism used according to the method for producing valine is a microorganism numbered CNCM I-5911, deposited on October 19, 2022 at the National Microbiological Collection Center of the Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

[0110] More preferably, the recovery of valine according to step b) comprises at least the following steps:

[0111] a) clarifying the fermentation medium to remove insoluble organic impurities,

[0112] b) treating the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities,

[0113] c) evaporating the water and crystallizing the product obtained,

[0114] d) recovering valine.

[0115] The skilled person is able to define the culture conditions of 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.

[0116] The process can be carried out in a batch process, in a fed-batch process or in a continuous process. It can be carried out under aerobic, microaerobic or anaerobic conditions or a combination thereof (e.g., aerobic conditions followed by anaerobic conditions).

[0117] "Under aerobic conditions" means that oxygen is provided to the culture by dissolving gases into the liquid phase. This can be accomplished by (1) bubbling an oxygen-containing gas (e.g., air) into the liquid phase or (2) shaking the container containing the culture medium to transfer oxygen contained in the headspace to the liquid phase. The main advantage of fermenting under aerobic conditions is that the presence of oxygen as an electron acceptor increases the ability of the strain to generate more energy in the form of ATP for cellular processes. Thus, the general metabolism of the strain is improved.

[0118] Microaerobic conditions are defined as culture conditions in which a low percentage of oxygen (eg, using a gas mixture containing between 0.1% and 10% oxygen, mixed with nitrogen to 100%) is dissolved into the liquid phase.

[0119] Anaerobic conditions are defined as culture conditions in which oxygen is not supplied to the culture medium. Strict anaerobic conditions are obtained by bubbling an inert gas such as nitrogen into the culture medium to remove trace amounts of other gases. Nitrates can be used as electron acceptors to improve ATP production by the strain and improve its metabolism.

[0120] The valine produced by the microorganism in the culture broth can be unambiguously determined by standard analytical means known to those skilled in the art. As non-limiting examples, valine can be quantified using isocratic HPLC (Pleissner et al., 2011) or nuclear magnetic resonance.

[0121] Example

[0122] The present invention is further defined in the following examples. It should be understood that although preferred embodiments of the present invention are indicated, these examples are provided by way of illustration only. It will be readily appreciated by those skilled in the art that these examples are not restrictive and that various modifications, substitutions, omissions and changes may be made without departing from the scope of the present invention.

[0123] method

[0124] The scheme used in the following examples is:

[0125] The present invention uses Solution 1 (chromosomal modification by homologous recombination, selection of recombinants and excision of the antibiotic cassette flanked by FRT sequences) and Solution 2 (Transduction of bacteriophage P1) has been fully described in patent application WO 2013 / 001055 (see in particular the "Example Schemes" section and Examples 1 to 8, incorporated herein by reference).

[0126] Scenario 3: Construction of recombinant plasmids.

[0127] Recombinant DNA technology is well described and well known to those skilled in the art. In brief, DNA fragments are subjected to PCR amplification using oligonucleotides (which can be defined by those skilled in the art) and using E. coli MG1655 genomic DNA or suitable synthetic fragments as a matrix. DNA fragments and selected plasmids are digested with compatible restriction enzymes (which can be defined by those skilled in the art), then connected and transformed into competent cells. Transformants are analyzed and the target recombinant plasmid is verified by DNA sequencing.

[0128] Solution 4: Evaluation of L-valine fermentation performance

[0129] The production of valine by the production strains was evaluated in the bioreactor using the medium MM_VAB10 and MM_VAB20 (Table 1) adjusted to pH 6.8. The MM_VAB10 medium was specifically used to monitor the ability of the strain to produce valine in the initial stage. The MM_VBA20 medium was specifically used to demonstrate the impact of genetic optimization under high valine content. 50 mL of pre-culture was added to a rich medium (with 5 g.L -1Glucose-free LB medium) at 30°C for 16 hours. This was used to inoculate a 200 mL culture to an OD of 0.5. 600 If necessary, antibiotics were added to the culture medium (final concentration was 50 mg / L -1 and 30mg.L -1 Spectinomycin and chloramphenicol). The temperature of the culture was 39°C. The culture was stopped when glucose was completely consumed within a maximum culture duration of 50 hours. After OPA / Fmoc derivatization, extracellular amino acids were quantified by HPLC, and other relevant metabolites (organic acids) were analyzed using HPLC with refractive index detection.

[0130] Table 1: Composition of MM_VAB10 and MM_VAB20 culture media

[0131]

[0132] In these cultures, the valine yield (YVal) is expressed as follows:

[0133]

[0134] And the valine productivity (PVal) is expressed as follows:

[0135]

[0136] Example 1: A valine-producing strain carrying more than one copy of the ilvA gene has improved valine production performance. Construction of a valine-producing strain with variable copy numbers of the ilvA gene

[0137] Construction of strain 1

[0138] According to schemes 1, 2 and 3, Escherichia coli MG1655 strain 1 was obtained in sequence:

[0139] - Knockout of lactate dehydrogenase (ldhA gene, SEQ ID NO: 9), alcohol dehydrogenase (adhE gene, SEQ ID NO: 11) and methylglyoxal synthase (mgsA gene, SEQ ID NO: 13),

[0140] - replacement of the acetohydroxyacid synthase I small regulatory subunit (ilvN gene, SEQ ID NO: 49) with the valine feedback resistance (FBR) protein (substitutions G20D, V21D and M22F of the IlvN*FBR protein (SEQ ID NO: 52) - Park et al., 2012) and overexpression by replacing the natural promoter with the artificial Ptrc promoter (Brosius et al., 1985),

[0141] - Overexpression of the following genes on the pCL1920 vector (Lerner & Inouye, 1990), organized in an operon under the control of the PR promoter together with the cI857 allele of the lambda phage thermosensitive repressor (SEQ ID NO: 80) (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994):

[0142] - vdh gene (SEQ ID NO:53), encoding valine dehydrogenase from Streptomyces aureofaciens (SEQ ID NO:54, Uniprot A0A1E7N3I8); more precisely, the vdh gene was artificially synthesized and its codon usage was optimized for Escherichia coli (SEQ ID NO:55).

[0143] - ilvD gene (SEQ ID NO:45), encoding IlvD dihydroxyacid dehydratase (SEQ ID NO:46),

[0144] - ilvC gene (SEQ ID NO:43), encoding IlvC keto-acid reductoisomerase (SEQ ID NO:44),

[0145] -ilvBN gene, which encodes two subunits of acetohydroxyacid synthase I, and ilvN*FBR allele (ilvB gene SEQ ID NO: 47; ilvN* gene SEQ ID NO: 51),

[0146] Generate plasmid 1.

[0147] This strain has one copy of the endogenous ilvA gene (SEQ ID NO: 1) encoding a threonine dehydratase (SEQ ID NO: 2).

[0148] Construction of strain 2

[0149] In order to construct strain 2, according to Scheme 1 and Scheme 2, another copy of the ilvA gene and its promoter are integrated into the pseudogene of strain 1, preferably the pseudogene cited in application US2012 / 0252077.

[0150] Construction of strain 3

[0151] To construct strain 3, the ilvA gene and its promoter were cloned into the pACYC plasmid (Bartolomé et al., 1991), generating plasmid 2, which was introduced into strain 1.

[0152] Improvement of valine production in strain 1 background related to ilvA gene copies

[0153] Strain 1 to strain 3 were cultured according to Protocol 4. Valine productivity and yield were measured.

[0154] Table 2: Valine productivity and yield of strains with one, two or more than two copies of the ilvA gene.

[0155]

[0156] The valine productivity and valine yield of strain 1 carrying one copy of the ilvA gene under MM_VAB10 medium conditions are referred to as "reference 1" and "reference 2", respectively, and the valine productivity and valine yield of strain 1 carrying one copy of the ilvA gene under MM_VAB20 medium conditions are referred to as "reference 3" and "reference 4", respectively. Compared to the appropriate reference, the symbol "≈" indicates an increase of less than 10%, the symbol "++" indicates an increase between 30% and 100%, and the symbol "+++" indicates an increase of more than 100%.

[0157] As shown in Table 2, regardless of the medium used, strain 2 carrying two copies of the ilvA gene had improved valine productivity compared to strain 1. And, especially under the MM_VAB20 medium condition, strain 3 carrying more than two copies of the ilvA gene had improved valine productivity and yield.

[0158] These results were unexpected, since in valine-producing strains described in the literature the ilvA gene is often deleted or attenuated.

[0159] Improvement of valine production in relation to ilvA gene copies in other valine-producing strain backgrounds

[0160] The beneficial effects of ilvA copies were demonstrated in other genetic backgrounds, more specifically:

[0161] - the W3110 E. coli strain described in Park et al., 2011 as "VAMF (pKBRilvBNmutCED, pTrc184ygaZHlrp)" and in which the ilvA gene is deleted, and

[0162] -W3110 E. coli strain described as “VHY18” in Hao et al., 2020, in which the ilvA gene has not been modified so that the strain carries one copy of the ilvA gene.

[0163] To increase the copy number of the ilvA gene in both strains to up to two, an additional copy of the ilvA gene and its promoter were added to the locus cited in application US2012 / 0252077 in both strains, and the original ilvA gene was reconstructed into the Park strain at the endogenous locus according to Schemes 1 and 2.

[0164] Park strain and Hao strain, as well as equivalent strains with two copies of the ilvA gene, were cultured according to the corresponding conditions described in Park et al., 2011 and Hao et al., 2020, and valine productivity was evaluated as described in Scheme 4.

[0165] In the Park strain background, the change of the ilvA gene from zero copies to two copies slightly improved productivity, while in the Hao strain background, the change of the ilvA gene from one copy to two copies improved productivity and yield by the same order of magnitude compared to strain 2 described in Example 1.

[0166] Furthermore, since there is no need to add leucine and isoleucine to the culture medium, reconstruction of the ilvA gene into the Park strain is economically advantageous.

[0167] Example 2: Punctual mutation in the argP gene improves the valine performance of a valine-producing strain carrying two copies of the ilvA gene

[0168] Construction of strain 4

[0169] To construct strain 4, according to Schemes 1 and 2, in strain 2, the wild-type argP allele encoding the DNA-binding transcriptional dual regulator (SEQ ID NO: 3) was replaced with the argP* allele encoding the ArgP mutant (SEQ ID NO: 5), wherein the ArgP mutant has an amino acid substitution of glutamic acid to aspartic acid at position 128 (SEQ ID NO: 6).

[0170] Improvement of valine production in strain 2 by the argP* allele

[0171] Strain 2 and strain 4 were cultured according to Protocol 4. Valine productivity and yield were measured.

[0172] Table 3: Valine productivity and yield of strains carrying in addition to 2 copies of the ilvA gene a mutation in the argP gene or not.

[0173]

[0174] The valine productivity and the valine yield of strain 2 carrying the wild-type form of the argP gene under MM_VAB10 medium conditions are referred to as "reference 5" and "reference 6", respectively, and the valine productivity and the valine yield of strain 2 under MM_VAB20 medium conditions are referred to as "reference 7" and "reference 8", respectively. Compared to the appropriate reference, the symbol "≈" indicates an increase of less than 10%, the symbol "++" indicates an increase between 30% and 100%, and the symbol "+++" indicates an increase of more than 100%.

[0175] As shown in Table 3, regardless of the medium used, strain 4 carrying a mutation in the argP gene (and 2 copies of the ilvA gene) had improved valine productivity compared to strain 2 carrying the wild-type allele of the argP gene. The mutation of argP did not affect the yield of valine.

[0176] Example 3: Point mutation in the fepA gene promoter improves the valine performance of a valine-producing strain carrying two copies of the ilvA gene

[0177] Construction of strain 5

[0178] The gene fepA (SEQ ID NO: 82) encodes the FepA protein (SEQ ID NO: 83), which is a ferric enterobactin outer membrane transporter. To construct strain 5, the wild-type fepA promoter sequence was replaced with a mutant promoter sequence in strain 2 according to Scheme 1 and Scheme 2. The mutant promoter has a nucleobase substitution of T to C at the -3 position from the start of transcription (SEQ ID NO: 8).

[0179] Improvement of valine production by strain 2 by mutations in the fepA promoter

[0180] Strain 2 and strain 5 were cultured according to Protocol 4. Valine productivity and yield were measured.

[0181] Table 4: Valine productivity and yield of strains carrying or not carrying in addition to the 2 copies of the ilvA gene a mutation in the fepA promoter.

[0182]

[0183]

[0184] The valine productivity and the valine yield of strain 2 carrying the wild-type form of the fepA promoter under MM_VAB10 medium conditions are referred to as "reference 5" and "reference 6", respectively, and the valine productivity and the valine yield of strain 2 under MM_VAB20 medium conditions are referred to as "reference 7" and "reference 8", respectively. The symbol "≈" indicates an increase of less than 10%, and "++" indicates an increase between 30% and 100%, compared to the appropriate reference.

[0185] As shown in Table 4, under MM_VAB20 medium conditions, strain 5 carrying a mutation in the fepA promoter (and 2 copies of the ilvA gene) had improved valine productivity compared to strain 2 carrying the wild-type sequence of the fepA promoter. The mutation of the fepA promoter did not affect the yield of valine.

[0186] Example 4: Point mutation in fepA promoter improves valine performance of valine producing strain carrying two copies of ilvA gene and a mutation in argP gene

[0187] Construction of strain 6

[0188] To construct strain 6, the wild-type fepA promoter sequence was replaced with a mutant promoter sequence in strain 4 according to Schemes 1 and 2. The mutant promoter had a nucleobase substitution of T to C at the -3 position from the start of transcription (SEQ ID NO: 8).

[0189] Improvement of valine production by strain 4 by mutations in the fepA promoter

[0190] Strain 4 and strain 6 were cultured according to Protocol 4. Valine productivity and yield were measured.

[0191] Table 5: Valine productivity and yield of strains carrying or not carrying, in addition to the 2 ilvA gene copies and the mutation in the argP gene, a mutation in the fepA promoter.

[0192]

[0193] The valine productivity and the valine yield of strain 4 carrying the argP mutant allele and the wild-type sequence of the fepA promoter under MM_VAB10 medium conditions are referred to as "reference 9" and "reference 10", respectively, and the valine productivity and the valine yield of strain 4 under MM_VAB20 medium conditions are referred to as "reference 11" and "reference 12", respectively. The symbol "≈" indicates an increase of less than 10%, and "++" indicates an increase between 10% and 30%, compared to the appropriate reference.

[0194] As shown in Table 5, under MM_VAB20 medium conditions, strain 6 carrying a mutation in the fepA promoter (and 2 copies of the ilvA gene and a mutation in the argP gene) had improved valine productivity compared to strain 4 carrying the wild-type sequence of the fepA promoter. The mutation of the fepA promoter did not affect the productivity of valine.

[0195] References

[0196] ·Bantscheff et al., (2007), Analytical and Bioanalytical Chemistry, vol.389(4):1017-1031.

[0197] ·Bartolomé B, Jubete Y, Martínez E, de la Cruz Fet al., (1991), Gene., 1991 Jun 15; 102(1):75-8.

[0198] ·Burnette, (1981), Analytical Biochemistry, 112(2):195-203.

[0199] ·Carrier T&Keasling J., (1999), Biotechnol Prog., 15(1):58-64. Construction and properties of a family of pACYC184-derived cloningvectors compatible with pBR322 and its derivatives.

[0200] ·Datsenko and Wanner, (2000), Proc Natl Acad Sci USA., 97: 6640-6645.

[0201] ·Davis&Olsen.,(2011),Mol.Biol.Evol.,28(1):211-221.

[0202] ·Deml et al., (2011), J. Virol., 75(22):10991-11001.

[0203] ·Engvall and Perlman(1981),Immunochemistry,8:871-874.Fed-batchculture of Escherichia coli for L-valine production based on in silico fluxresponse analysis.

[0204] ·Graf et al.,(2000),J.Virol.,74(22):10 / 22-10826.

[0205] ·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.,2020Nov;62:198-206.High-yield production of L-valinein engineered Escherichia coli by a novel two-stage fermentation.

[0206] ·Leuchtenberger,et al,(2005)Appl.Microbiol.Biotechnol.69,1-8

[0207] ·Needleman and Wunsch(1970),J.Mol.Biol.,48(3),443-453.

[0208] ·Park et al.,(2012),ACS synthetic biology,1(11):532-540

[0209] ·Park JH,Kim TY,Lee KH,Lee SYet al.,(2011),Biotechnol Bioeng.,2011Apr;108(4):934-46.

[0210] ·Salis H.,(2011),Methods Enzymol.,498:19-42

[0211] ·Segel I.,(1993),Enzyme kinetics,John Wiley&Sons,pp.44-54and 100-112。

Claims

1. A genetically modified microorganism for producing 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 the corresponding wild-type microorganism, and wherein the microorganism further comprises a mutant argP gene encoding a DNA-binding transcriptional dual regulator.

2. The microorganism according to claim 1, wherein the ilvA gene is overexpressed in the microorganism, such as by modifying a promoter regulating the expression of the ilvA gene, by increasing the number of copies of the ilvA gene present in the microorganism, or by overexpressing the ilvA gene from a plasmid, by increasing the stability of ilvA mRNA or by optimizing the ribosome binding site to increase the amount of IlvA protein.

3. The microorganism according to claim 1 or 2, wherein the ilvA gene is overexpressed in the microorganism by increasing the copy number of the ilvA gene present in the microorganism, preferably resulting in at least two copies of the gene, more preferably resulting in two copies of the gene.

4. The microorganism according to any one of claims 1 to 3, wherein the mutant argP gene is modified to result in a substitution of the amino acid at position 128, preferably a substitution of Glu by Asp.

5. The microorganism according to any one of claims 1 to 4, wherein the microorganism further overexpresses a fepA gene encoding a ferric ferrous enterobactin outer membrane transporter and / or exhibits increased ferric ferrous enterobactin outer membrane transporter activity compared to the expression level and / or ferric ferrous enterobactin outer membrane transporter activity in a corresponding wild-type microorganism.

6. The microorganism according to claim 5, wherein the fepA gene is overexpressed by genetic modification, such as by modifying a promoter regulating the expression of the fepA gene, by increasing the number of copies of the fepA gene present in the microorganism, or by overexpressing the fepA gene from a plasmid, by increasing the stability of fepA mRNA or by optimizing the ribosome binding site to increase the amount of FepA protein, preferably by mutating a promoter regulating the expression of the fepA gene. 7 . The microorganism according to claim 6 , wherein the fepA gene is overexpressed by making at least one base substitution in a promoter sequence regulating the expression of the fepA gene.

8. The microorganism according to any one of claims 1 to 7, further comprising a deletion of at least one gene selected from the group consisting of ldhA, adhE and mgsA.

9. The microorganism according to any one of claims 1 to 8, further comprising overexpression of at least one gene selected from the group consisting of vdh, ilvD, ilvC, ilvB and ilvN*.

10. The microorganism according to any one of claims 1 to 9, wherein the microorganism belongs to the bacterial family Enterobacteriaceae, Corynebacteriaceae, Bacillaceae, Streptococcae or Lactobacillae, or to the fungal family, such as Hemiascomycetus, filamentous fungi or yeast.

11. The microorganism according to claim 10, wherein the Enterobacteriaceae bacterium is Escherichia coli, the Corynebacterium family bacterium is Corynebacterium glutamicum, or the Bacillaceae family is Bacillus subtilis, the Streptococcus family is Streptococcus thermophiles, the Lactobacillaceae family is Lactobacillus lactis, the Hemiascomycete yeast is Saccharomyces cerevisiae or Yarrowia lipolytica, and the filamentous fungus is Trichoderma rezeii or Aspergillus niger, preferably wherein the microorganism is Escherichia coli.

12. The microorganism according to claim 1, numbered CNCM I-5911, deposited on October 19, 2022 at the National Collection of Microorganisms of the Pasteur Institute, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.

13. A method for producing valine, the method comprising the following steps: a) cultivating the microorganism genetically modified for producing valine according to any one of claims 1 to 12 in a suitable culture medium comprising a carbon source, and b) recovering valine from the culture medium.

14. The method according to claim 13, wherein the recovery of valine comprises at least the following steps: a) clarifying the fermentation medium to remove insoluble organic impurities, b) treating the product of the previous step on an adsorbent such as activated carbon to remove soluble organic and inorganic impurities, c) evaporating the water and crystallizing the product obtained, d) recovering valine.

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