Microorganisms and methods for improving leucine and / or isoleucine production

Abstract

The present invention relates to a microorganism genetically modified against the production of leucine and / or isoleucine, wherein the microorganism comprises the expression of a heterologous gapN gene encoding an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and a reduction in the expression of the gapA and gltA genes as compared to the unmodified microorganism. The invention also relates to a method for producing leucine and / or isoleucine using said microorganism.

Description

Technical Field

[0001] The present invention relates to microorganisms genetically modified for improved production of leucine and / or isoleucine, and to methods for improving leucine and / or isoleucine production using said microorganisms. Background Art

[0002] Amino acids are used in many industrial fields, including food, animal feed, cosmetics, pharmaceutical and chemical industries, and have an estimated annual global market growth rate of 5% to 7% (Leuchtenberger et al., 2005). Among them, leucine and isoleucine are particularly important for the nutrition of humans and many livestock species because they are essential amino acids that cannot be synthesized by mammals. Therefore, they are commonly used as food additives and in dietary supplements, where leucine is also used as a flavor enhancer. Leucine and isoleucine also play a role as precursors in the synthesis of antibiotics (such as polyketides).

[0003] Leucine and isoleucine can be produced via chemosynthesis, extraction from protein hydrolysate or microbial fermentation. In these technologies, due to relevant economic and environmental advantages, fermentation is the most commonly used today. Especially, fermentation provides a useful way of using abundant, renewable and / or cheap materials as main carbon source. In addition, although when using chemosynthesis, produce equimolar amount of D-enantiomer and L-enantiomer, thereby need the other downstream separation of L-enantiomer, fermentation only produces L-enantiomer. The biosynthesis that leucine and isoleucine are carried out by fermentation uses the microorganism of Corynebacterium (Corynebacterium) or Escherichia (Escherichia), as Corynebacterium glutamicum (Corynebacterium glutamicum) or Escherichia coli (Escherichia coli) to carry out.

[0004] Initially, strains producing leucine and isoleucine were isolated by random mutagenesis. However, recently, rational metabolic engineering has been performed on microorganisms, where strategies to improve amino acid production have focused on removing feedback inhibition, improving upstream central carbon flux, and reducing downstream synthesis of undesirable byproducts (see, e.g., Yamamato et al., 2017; Park et al., 2010).

[0005] As an example, for isoleucine, amino acid production can be improved by incorporating anti-feedback threonine dehydratase and aspartate kinase III (encoded by ilvA and lysC, respectively, in E. coli), while leucine production can be improved by removing feedback inhibition of leuA. As a further example, production can be improved by overexpressing the leuE gene encoding L-leucine specific export protein in E. coli or deleting the livK gene encoding L-leucine specific transporter (Park et al., 2010).

[0006] In view of the growing demand for leucine and isoleucine in industrial applications, there is still a need to further improve the production of these amino acids. In particular, there is still a need for improved microorganisms that can produce leucine or isoleucine with high levels of productivity, titer and yield, particularly from cheap and / or abundant carbon sources (such as glucose). There is also a need for improved methods for producing leucine or isoleucine on an industrial scale, ideally, wherein the productivity, titer and yield of leucine or isoleucine are at least similar to those obtained with current methods. Summary of the invention

[0007] The present invention solves the above demand, provides a kind of microorganism that carries out genetic modification for the generation of leucine and / or isoleucine and a kind of method using said microorganism to produce leucine and / or isoleucine.Compared with unmodified microorganism, the microorganism that carries out genetic modification for the generation of leucine and / or isoleucine significantly expresses the heterologous gapN gene of coding NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and has the expression of the weakening of gapA of coding glyceraldehyde-3-phosphate dehydrogenase A and gltA of coding citrate synthase.In fact, the inventors have found that the generation of leucine or isoleucine that improves is advantageously shown by such microorganism, because productivity, titer and yield increase.

[0008] Preferably, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase encoded by the gapN gene has at least 80% identity with GapN from Streptococcus mutans.

[0009] Preferably, the gapA gene is deleted.

[0010] Preferably, the microorganism further comprises an attenuation of the expression of the gapB and / or gapC gene, preferably a deletion of the gapB and gapC gene, compared to an unmodified microorganism.

[0011] Preferably, the microorganism further comprises overexpression of at least one gene selected from ackA, pta and acs compared to an unmodified microorganism.

[0012] Preferably, the microorganism is genetically modified for the production of leucine and comprises overexpression of the following genes compared to the unmodified microorganism: ilvBN, ilvC, ilvD, leuA*, leuB, leuC, leuD and ilvE.

[0013] Preferably, the microorganism is genetically modified for the production of isoleucine and comprises:

[0014] a) Expression of heterologous cimA* genes,

[0015] b) overexpression of the following genes compared to the unmodified microorganism: ilvIH*, ilvC, ilvD, leuB, leuC, leuD and ilvE, and

[0016] c) Attenuation of the leuA gene.

[0017] Preferably, compared to an unmodified microorganism, the microorganism further comprises:

[0018] a) attenuation of the expression of at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda, and gnd, and / or

[0019] b) overexpression of at least one gene selected from the group consisting of pntAB, gdhA, leuE and ygaZH.

[0020] Preferably, in the microorganism, at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda and gnd is deleted.

[0021] Preferably, the microorganism belongs to the genus Escherichia, more preferably wherein the microorganism is Escherichia coli (E. coli); the genus Corynebacterium, more preferably wherein the microorganism is Corynebacterium glutamicum (C. glutamicum); or the genus Streptococcus (Streptococcus), more preferably wherein the microorganism is selected from Streptococcus thermophilus (S. thermophilus) and Streptococcus salivarius (S. salivarius); most preferably wherein the microorganism is Escherichia coli.

[0022] The present invention further relates to a method for producing leucine and / or isoleucine, the method comprising the steps of:

[0023] a) culturing a microorganism genetically modified for the production of leucine and / or isoleucine as provided herein in a suitable medium comprising a carbon source, and

[0024] b) recovering leucine and / or isoleucine from the culture medium.

[0025] Preferably, the culture medium further comprises acetate.

[0026] Preferably, the carbon source is glucose, fructose, galactose, lactose and / or sucrose.

[0027] Preferably, step b) of the process comprises a crystallization step. DETAILED DESCRIPTION

[0028] 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 illustrated, and certainly can change.In fact, can carry out various modifications, substitutions, omissions and changes without departing from the scope of the present invention.It should also be understood that the term used herein is only used to describe the purpose of a specific embodiment of the present invention, and is not intended to be restrictive.

[0029] Whether above or below, all publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. In addition, unless otherwise indicated, the practice of the present invention adopts conventional microbiology and molecular biology techniques within the technical scope of the art. Such techniques are well known to those skilled in the art and are fully explained in the literature (see, for example, Prescott et al. (1999) and Sambrook and Russell (2001)).

[0030] 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 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.

[0031] 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, etc.

[0032] The terms "comprise", "contain", "include" and variations such as "comprising" are used herein in an inclusive sense, i.e. to specify the presence of stated features in various embodiments of the invention but do not preclude the presence or addition of additional features.

[0033] A first aspect of the present invention relates to a microorganism genetically modified for the production of leucine and / or isoleucine. As used herein, the term "microorganism" refers to a living microscopic organism, which can be a unicellular or multicellular organism and can be found generally in nature. Microorganisms provided herein are preferably bacteria. Preferably, microorganisms are selected from Enterobacteriaceae, Streptococcaceae or Corynebacteriaceae. More preferably, microorganisms are species of Escherichia, Streptococcus or Corynebacterium. Even more preferably, the Enterobacteriaceae bacterium is Escherichia coli, the Streptococcus bacterium is Streptococcus thermophilus or Streptococcus salivarius, and the Corynebacterium bacterium is Corynebacterium glutamicum. Most preferably, microorganisms are Escherichia coli.

[0034] The terms "recombinant microorganism", "genetically modified 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 genetic engineering or by adaptation, wherein the microorganism is cultured under conditions where a specific stress is imposed on the microorganism and mutagenesis is induced; and / or by forcing metabolic pathways to develop and evolve by combining directed mutagenesis and directed evolution under specific selection pressures.

[0035] The microorganism that carries out genetic modification for the leucine and / or isoleucine generation of increase means that the microorganism is following recombinant microorganism, and it has the leucine and / or isoleucine generation of increase compared with the parent microorganism that does not comprise genetic modification.In other words, compared with the corresponding unmodified microorganism, the microorganism has been carried out genetic modification for the leucine and / or isoleucine generation of increase.

[0036] Microorganisms can be modified in particular to regulate the expression level of endogenous genes or the production level of corresponding proteins or the activity of corresponding enzymes. 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 or introducing heterologous sequences to replace endogenous regulatory elements in addition to endogenous regulatory elements. Endogenous genes can also be overexpressed by introducing one or more supplementary copies of genes into chromosomes or onto plasmids. In this case, the endogenous genes originally present in the microorganism may be deleted. It is also possible to increase or reduce the activity of endogenous gene expression levels, protein production levels or encoded proteins by introducing mutations into the coding sequence of the gene or into non-coding sequences. When no modification is performed 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.

[0037] 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)), or DNA reorganization or fallibility PCR or use the culture conditions that microorganisms are applied to specific stress and induce mutagenesis to introduce. In the region positioned at the upstream of the gene, inserting one or more supplementary nucleotides can especially regulate gene expression.

[0038] One particular way to regulate the expression of an endogenous gene is to swap the endogenous promoter of the gene (e.g., the 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 well within the capabilities of those skilled in the art to select an appropriate promoter to regulate the expression of an endogenous gene. Such promoters are, for example, the Ptrc, Ptac, Ptet or Plac promoters, or the λP L (PL) or λP R (PR) Promoter. A promoter may be "inducible" by specific compounds or by specific external conditions such as temperature or light or small molecules such as antibiotics.

[0039] 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 above methods. Non-synonymous amino acid mutations present in a transcription factor may significantly change the binding affinity of the transcription factor to a cis-element, change the binding of a ligand to the transcription factor, etc.

[0040] Microorganism can also be genetically modified to express one or more foreign or heterologous genes, so as to overexpress the corresponding gene product (for example, enzyme). As used herein, "foreign" or "heterologous" gene refers to a gene encoding a protein or polypeptide, and the gene is introduced into a microorganism that is not naturally present in the gene. In the context of the present invention, gapN and cimA genes are especially heterologous genes. In particular, heterologous genes can be directly integrated into the chromosome of a microorganism, or are expressed extrachromosomally in a microorganism by a plasmid or a vector. In order to successfully express, one or more heterologous genes must be introduced into a microorganism together with all the necessary regulatory elements for their expression, or one or more foreign genes are introduced into a microorganism that has already contained all the necessary regulatory elements for their expression. It is a routine task for those skilled in the art to genetically modify or transform a microorganism with one or more foreign genes.

[0041] The given heterologous gene of one or more copies can be introduced on chromosome by methods well known in the art (such as by genetic recombination). When gene is expressed outside chromosome, it can be carried by plasmid or vector. Different types of plasmids are especially available, and they may be different in replication origin and / or copy number in cell. For example, the microorganism transformed by plasmid can contain 1 to 5 copies of plasmid, about 20 copies or even up to 500 copies, depending on the property of selected plasmid. A variety of plasmids with different replication origins and / or copy numbers are well known in the art and can be easily selected for such purposes by skilled practitioners, including for example pTrc, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236 or pCL1920.

[0042] It should be understood that in the context of the present invention, when a heterologous gene encoding a protein of interest is expressed in a microorganism (e.g., E. coli), a synthetic form of the gene is preferably constructed by replacing non-preferred codons or less preferred codons with preferred codons encoding the same amino acid in the microorganism. In fact, it is well known in the art that codon selection differs between microbial species, and this may affect the level of recombinant production of the protein of interest. In order 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 and Olsen (2011). In particular, several software programs have been developed for codon optimization determination, such as Software (Lifetechnologies) or OptimumGene TM Software (GenScript). In other words, the heterologous gene encoding the protein of interest is preferably codon optimized for production in the selected microorganism. As a specific example, the heterologous gapN gene can be codon optimized for expression in a microorganism such as (Escherichia coli).

[0043] Based on a given amino acid sequence, the skilled artisan is also able to identify appropriate polynucleotides encoding the polypeptide (e.g., in available databases such as Uniprot), or synthesize the corresponding polypeptide or polynucleotides encoding the polypeptide. De novo synthesis of polynucleotides can be performed, for example, by initially synthesizing individual nucleic acid oligonucleotides and hybridizing these oligonucleotides with oligonucleotides complementary thereto so that they form double-stranded DNA molecules, and then ligating the individual double-stranded oligonucleotides to obtain the desired nucleic acid sequence.

[0044] The terms "production", "overproduction" or "overproduction" of a protein of interest (e.g., an enzyme) herein refer to an increase in the level of production and / or activity of the protein in a microorganism compared to a corresponding parent microorganism that does not comprise the modifications present in the genetically modified microorganism (i.e., in an unmodified microorganism). The heterologous gene or protein may be considered to be "expressed" or "overexpressed" and "produced" or "overproduced", respectively, in a genetically modified microorganism when compared to a corresponding parent microorganism in which the heterologous gene or protein is not present. In contrast, the terms "attenuating" or "attenuation" of the synthesis of a protein of interest refer to a decrease in the level of production and / or activity of the protein in a microorganism compared to the parent microorganism. Similarly, "attenuation" of gene expression refers to a decrease in the level of gene expression compared to the parent microorganism. The reduction of expression may be due in particular to the exchange of a wild-type promoter for 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). The complete reduction of the production level and / or activity of the target protein means that the production and / or activity are eliminated; therefore, the production level of the protein is zero. The complete reduction of the production level and / or activity of the target protein may be due to the complete inhibition of gene expression. This inhibition may be the inhibition of gene expression, the deletion of all or part of the promoter region necessary for gene expression, or the deletion of all or part of the 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, the inhibition of gene expression may be achieved by homologous recombination techniques, which are well known to those skilled in the art (Datsenko and Wanner, 2000).

[0045] Thus, regulating the production 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 and / or by introducing one or more heterologous genes encoding one or more of said proteins into the microorganism.

[0046] As used herein, the term "production 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 production level of the protein encoded by the gene can be measured by, for example, SDS-PAGE, HPLC, LC / MS and other quantitative proteomics techniques (Bantscheff et al., 2007); or, when antibodies to the protein are available, 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 quantified by, for example, Southern blotting, fluorescent in situ hybridization (FISH), qPCR, etc. using probes based on gene sequences after restriction of chromosomal DNA.

[0047] Overexpression of a given gene or overproduction of the corresponding protein can be verified by comparing the expression level of the gene or the synthesis level of the protein in the genetically modified organism with the expression level of the same gene or the synthesis level of the same protein in a control microorganism without the genetic modification (i.e., the parent strain or an unmodified microorganism).

[0048] The microorganisms provided herein that are genetically modified for the production of leucine and / or isoleucine comprise

[0049] - a heterologous enzyme having NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity, and

[0050] - Reduction in the activity of glyceraldehyde-3-phosphate dehydrogenase A (GapA) and citrate synthase (GltA) compared to unmodified microorganisms.

[0051] Indeed, the inventors have shown that these genetic modifications advantageously improve leucine and isoleucine titers, productivity and yield compared to microorganisms not comprising the above modifications.

[0052] The "activity" or "function" of an enzyme refers to the reaction catalyzed by the enzyme for converting its 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, incorporated herein by reference, particularly at pages 44-54 and 100-112.

[0053] The enzyme with NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity can be a phosphorylase or a non-phosphorylase. It is preferably GapN. GapN can be of bacterial, archaeal or eukaryotic origin. Preferably, GapN is of bacterial origin. GapN can be one of those described in Figure 4 of Iddar et al., 2005, which is incorporated herein by reference. In particular, the GapN enzyme can be from a species of Streptococcus (e.g., from mutans Streptococcus (S.mutans), Streptococcus pyogenes (S.pyogenes)), a species of Bacillus (Bacillus) (e.g., Bacillus cereus (B.cereus), Bacillus licheniformis (B.licheniformis), Bacillus thuringiensis (B.thuringiensis)), a species of Clostridium (e.g., C.acetobutylicum) or from pea (Pisum savitum). Preferably, the GapN enzyme is from Streptococcus mutans, Streptococcus pyogenes, Clostridium acetobutylicum, Bacillus cereus or pea, more preferably from Streptococcus mutans. GapN preferably has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with a GapN enzyme having a sequence of SEQ ID NO:36, 38, 40, 42 or 44. More preferably, GapN has a sequence of SEQ ID NO:36. GapN can be a functional variant or functional fragment of one of the GapN enzymes described herein. The corresponding gapN gene encoding GapN preferably has at least 80%, 90%, 95% or 100% sequence identity with SEQ ID NO:35, 37, 39, 41 or 43 (more preferably SEQ ID NO:35).

[0054] As used herein, the "functional fragment" of an enzyme refers to a portion of the amino acid sequence of an enzyme that at least comprises all regions necessary for the biological activity of the enzyme. These portions of the sequence can have various lengths, provided that the biological activity of the amino acid sequence of the reference enzyme is retained by the portion. In other words, the functional fragment of an enzyme as provided herein has enzymatic activity.

[0055] As used herein, "functional variant" refers to a protein that is structurally different from the amino acid sequence of a reference protein but generally retains all the necessary functional characteristics of the reference protein. Variants of a protein may be naturally occurring variants or non-naturally occurring variants. Such non-naturally occurring variants of a reference protein may be prepared, for example, by mutagenesis techniques of the encoding nucleic acid or gene, such as by random mutagenesis or site-directed mutagenesis.

[0056] Structural differences can be limited in such a way that the amino acid sequence of the reference protein and the amino acid sequence of the variant can be closely similar in general, and identical in many regions. Structural differences may be caused by conservative or non-conservative amino acid substitutions, deletions and / or additions between the amino acid sequence of the reference protein and the variant. The only proviso is that even if some amino acids are substituted, deleted and / or added, the biological activity of the amino acid sequence of the reference protein is still retained by the variant. As a non-limiting example, this variant of GapN retains its NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity. The ability of this activity can be demonstrated according to in vitro test assessment variants known to those skilled in the art. It should be noted that compared with the activity of the amino acid sequence of the reference enzyme provided herein (for example, the gene / enzyme of the specific microbial species provided herein or with the specific sequence provided as in the corresponding SEQ ID NO), the activity of the variant may be different in efficiency.

[0057] " Functional variant " of enzyme as described herein includes but is not limited to the enzyme with at least 60% similar or identical amino acid sequence after the amino acid sequence alignment of the enzyme as provided herein with coding. According to the present invention, this variant preferably has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence similarity or identity with protein as described herein. Said functional variant also has the same enzyme function as enzyme provided herein. As non-limiting example, the functional variant of GapN of SEQ ID NO:36 has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with said sequence. As a non-limiting example, the means for determining sequence identity are further provided below.

[0058] Preferably, the attenuation of GapA and GltA activity is caused by the inhibition of expression of gapA and gltA genes compared to unmodified microorganisms. The activity of GapA and / or GltA enzymes can be completely attenuated. Complete attenuation is preferably due to the partial or complete deletion of the gene encoding the enzyme. Preferably, GapA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:34. Preferably, the gapA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO:33. Preferably, the gapA gene is deleted. Preferably, GltA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:32. Preferably, the gltA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO:31.

[0059] The microorganism genetically modified for the production of leucine and / or isoleucine of the present invention preferably comprises

[0060] - expression of a heterologous gapN gene encoding a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and

[0061] - Attenuation of the expression of the gapA and gltA genes compared to an unmodified microorganism.

[0062] In addition to the modifications described above, the microorganism genetically modified for the production of leucine and / or isoleucine may comprise one or more additional modifications of the modifications described below.

[0063] In particular, the microorganism may further comprise a weakening of D-erythrose-4-phosphate dehydrogenase (GapB) activity. Preferably, the production of GapB is partially or completely weakened. Preferably, GapB has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to the sequence of SEQ ID NO:46. Preferably, the weakening of GapB activity is caused by the inhibition of expression of the gapB gene encoding the enzyme. Preferably, the weakening of expression is caused by the partial or complete deletion of the gapB gene. Preferably, the gapB gene has at least 80%, 90%, 95% or 100% sequence identity to the sequence of SEQ ID NO:45.

[0064] The microorganism may further comprise a weakening of glyceraldehyde-3-phosphate dehydrogenase (GapC) activity. Preferably, the production of GapC is partially or completely weakened. Preferably, GapC has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to the sequence of SEQ ID NO:125, 127 or 129. Preferably, the weakening is caused by the inhibition of expression of the gapC gene encoding the enzyme. Preferably, the weakening of expression is caused by the partial or complete deletion of the gapC gene. In some microorganisms, the gapC gene is a pseudogene. Therefore, as used herein, "gapC" can refer to a functional gene or a pseudogene. The gapC pseudogene or functional gene preferably has at least 80%, 90%, 95% or 100% sequence identity to the sequence of SEQ ID NO:47, 124, 126 or 128. In the case where gapC is a pseudogene, the pseudogene advantageously lacks to avoid the pseudogene from returning to a functional gene.

[0065] Preferably, the microorganism comprises an attenuation of the expression of the gapB gene and a deletion of the gapC pseudogene, more preferably a deletion of the gapB and gapC genes, compared to an unmodified microorganism.

[0066] The microorganism genetically modified for the production of leucine and / or isoleucine preferably further comprises an increased activity of at least one of the following enzymes compared to an unmodified microorganism: acetate kinase (AckA), phosphate acetyltransferase (Pta) and acetyl-CoA synthetase (Acs). Preferably, the microorganism comprises an overproduction of at least one protein selected from AckA, Pta and Acs compared to an unmodified microorganism. More preferably, the Pta protein is overproduced compared to an unmodified microorganism.

[0067] Preferably, AckA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 83. Preferably, Pta has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 61. Preferably, Acs has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 85.

[0068] Preferably, the overproduction of the one or more proteins is caused by overexpression of a gene encoding the protein (i.e., at least one of the genes selected from ackA, pta and acs). Preferably, the ackA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 82. Preferably, the pta gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 60. Preferably, the acs gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 84.

[0069] Preferably, the microorganism comprises overexpression of at least one gene selected from ackA, pta and acs compared to an unmodified microorganism, more preferably comprises overexpression of the pta gene compared to an unmodified microorganism.

[0070] Compared to an unmodified microorganism, the microorganism used to produce leucine may comprise an increase in the activity of at least one of the following enzymes: acetohydroxyacid synthase I (IlvBN), ketol-acid reductoisomerase (NADP(+)) (IlvC), dihydroxyacid dehydratase (IlvD), 2-isopropylmalate synthase (LeuA*), 3-isopropylmalate dehydrogenase (LeuB), 3-isopropylmalate dehydratase (LeuCD) and branched-chain amino acid transaminase IlvE. Preferably, the microorganism used to produce leucine comprises an overproduction of at least one of the following proteins: IlvBN, IlvC, IlvD, LeuA*, LeuB, LeuC, LeuD and IlvE. LeuA* is a feedback-resistant (FBR) protein. Acetohydroxyacid synthase I may also be feedback-resistant (IlvBN*).

[0071] As used herein, the term "anti-feedback protein" refers to a protein that has been modified such that feedback inhibition (ie, reduction in enzyme activity mediated by binding of a product to the enzyme) of the protein is reduced or even eliminated.

[0072] Preferably, IlvB and IlvN have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO:8 and 10, respectively. When IlvN* is overproduced instead of IlvN, the protein preferably has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:12. When compared to SEQ ID NO:10, IlvN* comprises the substitutions G20D, V21D and M22F. Preferably, IlvC has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:20. Preferably, IlvD has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:18. Preferably, LeuA*, LeuB, LeuC and LeuD have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 24, 26, 28 and 30, respectively. When compared to SEQ ID NO: 22, LeuA* comprises the substitution G462D. Preferably, IlvE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 16.

[0073] Preferably, the overproduction of the one or more proteins is caused by overexpression of genes encoding said proteins, i.e., ilvBN (or ilvBN*), ilvC, ilvD, leuA*, leuB, leuC and / or leuD genes. Preferably, the ilvB and ilvN genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NOs: 7 and 9, respectively. Preferably, the ilvN* gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 11, wherein ilvN* encodes a protein having the substitutions G20D, V21D and M22F relative to the wild-type protein having the sequence SEQ ID NO: 10. Preferably, the ilvC gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 19. Preferably, the ilvD gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 17. Preferably, the leuA*BCD genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 23, 25, 27 and 29, respectively, wherein the leuA* gene encodes a protein having the substitution G462D relative to the wild-type protein having the sequence of SEQ ID NO: 22. Preferably, the ilvE gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 15.

[0074] Preferably, the microorganism is genetically modified for the production of leucine and comprises overexpression of the following genes compared to the unmodified microorganism: ilvBN, ilvC, ilvD, leuA*, leuB, leuC, leuD and ilvE.

[0075] Preferably, overexpression occurs by replacing the natural promoter with an artificial promoter (such as the Ptrc promoter). Alternatively, a vector (e.g., a pCL1920 vector) containing one or more genes under the control of a strong promoter or an inducible promoter may be introduced into the microorganism and the one or more genes may be overexpressed.

[0076] Preferably, the microorganism is genetically modified for the production of isoleucine and comprises:

[0077] - expression of a heterologous enzyme having citramalate synthase activity,

[0078] - an increased activity of at least one of the following enzymes compared to an unmodified microorganism: acetolactate synthase III (IlvIH*), IlvC, IlvD, LeuB, LeuCD and IlvE, and

[0079] -Reduction in 2-isopropylmalate synthase (LeuA) activity.

[0080] IlvH* is a FBR protein.

[0081] The heterologous enzyme having citramalate synthase activity preferably has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the CimA enzyme having the sequence of SEQ ID NO: 75. Preferably, the heterologous enzyme having citramalate synthase activity is CimA from Methanocaldococcus jannaschii or a functional fragment or functional variant thereof, more preferably an anti-feedback functional variant thereof (CimA*). Preferably, the FBR citramalate synthase has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the CimA enzyme having the sequence of SEQ ID NO:77, and where the sequence is not 100% identical to SEQ ID NO:77, it has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with CimA* comprising substitutions I47V, E114V, H126Q, T204A, L238S and V373STOP.

[0082] The cimA gene preferably encodes a CimA enzyme having at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to SEQ ID NO: 75. The cimA* gene preferably encodes a CimA* enzyme having at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to SEQ ID NO: 77. Preferably, the cimA gene has at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 74. Preferably, the cimA* gene has at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO: 76, more preferably wherein in the case where the sequence is not 100% identical to the sequence of SEQ ID NO: 76, the cimA* gene encodes a protein having substitutions I47V, E114V, H126Q, T204A, L238S and V373STOP.

[0083] Preferably, the microorganism for producing isoleucine comprises an overproduction of at least one of the following proteins: IlvIH*, IlvC, IlvD, LeuB, LeuCD and IlvE. Preferably, IlvH* has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 69 or 71, respectively, wherein IlvH* comprises the substitutions G14D and S17F compared to SEQ ID NO: 67, or comprises the substitutions N29K and Q92STOP when compared to SEQ ID NO: 67. Preferably, IlvC has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 20. Preferably, IlvD has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 18. Preferably, LeuB, LeuC and LeuD have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO: 26, 28 and 30, respectively. Preferably, IlvE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 16.

[0084] Preferably, the overproduction of said one or more proteins is caused by overexpression of the genes encoding said proteins, i.e. the ilvIH*, ilvC, ilvD, leuC, leuD, leuB and / or ilvE genes.

[0085] Preferably, the ilvI gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 72. Preferably, the ilvH* gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 68 or 70, wherein in the case where the sequence is not 100% identical to the sequence of SEQ ID NO: 68 or 70, respectively, the ilvH* gene encodes a protein having the substitutions G14D and S17F or the substitutions N29K and Q92STOP. Preferably, the ilvC gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 19. Preferably, the ilvD gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 17. Preferably, the leuB, leuC and leuD genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NOs: 25, 27 and 29, respectively. Preferably, the ilvE gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 15. Preferably, the microorganism is genetically modified for the production of isoleucine and comprises overexpression of the following genes compared to the unmodified microorganism: ilvIH*, ilvC, ilvD, leuC, leuD, leuB and / or ilvE genes.

[0086] Preferably, LeuA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 22. Preferably, the expression of LeuA is partially or completely attenuated. Preferably, the attenuation of LeuA activity is caused by inhibition of expression of the leuA gene encoding the enzyme. Preferably, the attenuation of expression is caused by partial or complete deletion of the leuA gene. Preferably, the leuA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 21.

[0087] Preferably, the microorganism is genetically modified for the production of isoleucine and comprises:

[0088] - Expression of heterologous cimA* genes,

[0089] - Overexpression of the following genes compared to the unmodified microorganism: ilvIH*, ilvC, ilvD, leuC, leuD, leuB and ilvE, and

[0090] - Attenuation of the leuA gene.

[0091] Preferably, the overexpression of endogenous genes occurs by replacing the natural promoter with an artificial promoter (such as the Ptrc promoter). Alternatively, a vector (e.g., a pCL1920 vector) containing one or more genes under the control of a strong promoter or an inducible promoter can be introduced into the microorganism, and the one or more genes are overexpressed.

[0092] Preferably, when the protein is an endogenous protein, one or more of any of the above FBR proteins replace the corresponding wild-type protein in the microorganism (e.g., IlvH* replaces the wild-type IlvH in the microorganism). As a non-limiting example, the wild-type protein can be replaced by an FBR mutant by deleting the gene encoding the wild-type protein in the microorganism and incorporating a gene encoding the FBR mutant (e.g., by transforming the microorganism with a plasmid that overexpresses the gene), or directly mutating the wild-type gene present in the microorganism so that it becomes feedback resistant.

[0093] Preferably, the microorganism is genetically modified for the production of leucine or isoleucine and further comprises, compared to an unmodified microorganism:

[0094] - attenuation of overproduction of at least one of the following proteins: soluble pyridine nucleotide transhydrogenase (UdhA), pyruvate dehydrogenase (AceEF), 2-oxoglutarate dehydrogenase (SucAB), pyruvate oxidase (PoxB), branched-chain amino acid transport system 2 carrier protein (BrnQ), branched-chain amino acid / phenylalanine transport system (LivKHMGF), lactate dehydrogenase (LdhA), alcohol dehydrogenase (AdhE), methylglyoxal synthase (MgsA), fumarate reductase complex (FrdABCD), pyruvate formate lyase (PflAB), glucose-6-phosphate 1-dehydrogenase (Zwf), phosphogluconate dehydratase (Edd), KHG / KDPG aldolase (Eda) and 6-phosphogluconate dehydrogenase (Gnd), and / or

[0095] - Increased expression of at least one of the following proteins: NAD(P) transhydrogenase (PntAB), glutamate dehydrogenase (GdhA), leucine exporter (LeuE) and valine exporter (YgaZH).

[0096] Preferably, UdhA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:49. Preferably, AceE and AceF have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO:51 and 53, respectively. Preferably, SucA and SucB have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NO:57 and 59, respectively. Preferably, PoxB has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:55. Preferably, BrnQ has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:87. 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:89, 91, 93, 95 and 97, respectively. Preferably, LdhA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:2. Preferably, AdhE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:4. Preferably, MgsA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO:107. 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 NOs: 99, 101, 103 and 105, respectively. Preferably, PflA and PflB have at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequences of SEQ ID NOs: 109 and 111, respectively. Preferably, Zwf has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 113. Preferably, Edd has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 115. Preferably, Eda has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 117. Preferably, Gnd has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity to the sequence of SEQ ID NO:119.

[0097] Preferably, the attenuation of expression is caused by the partial or complete deletion of the gene encoding the protein (i.e., at least one of the udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda and gnd genes).

[0098] Preferably, the udhA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO:48. Preferably, the aceEF gene has at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO:50 and 52, respectively. Preferably, the sucAB gene has at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO:56 and 58, respectively. Preferably, the poxB gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO:54. Preferably, the brnQ gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO:86. Preferably, the livKHMGF gene has at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NOs: 88, 90, 92, 94 and 96, respectively. Preferably, the ldhA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 1. Preferably, the adhE gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 3. Preferably, the mgsA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 106. Preferably, the frdABCD genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NOs: 98, 100, 102 and 104, respectively. Preferably, the pflAB gene has at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 108 and 110, respectively. Preferably, the zwf gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 112. Preferably, the edd gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 114. Preferably, the eda gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 116. Preferably, the gnd gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 118. Preferably, at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda and gnd is deleted. Preferably, the genes udhA, aceEF, sucAB and poxB are attenuated, more preferably deleted, compared to an unmodified microorganism.

[0099] Preferably, PntAB has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 121 (PntA) and 123 (PntB). Preferably, GdhA has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 63. Preferably, LeuE has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 65. Preferably, YgaZH has at least 80%, 90%, 95% or 100% sequence similarity or sequence identity with the sequence of SEQ ID NO: 79 (YagZ) and 81 (YagH). Preferably, GdhA and LeuE proteins are overexpressed compared to the unmodified microorganism.

[0100] Preferably, the overproduction of the one or more proteins is caused by overexpression of genes encoding the proteins, i.e., at least one of the pntAB, gdhA, leuE and ygaZH genes. Preferably, the pntAB genes have at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 120 (pntA) and 122 (pntB). Preferably, the gdhA gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 62. Preferably, the leuE gene has at least 80%, 90%, 95% or 100% sequence identity with the sequence of SEQ ID NO: 64. Preferably, the ygaZH gene has at least 80%, 90%, 95% or 100% sequence identity with the sequences of SEQ ID NO: 78 (ygaZ) and SEQ ID NO: 80 (ygaH).

[0101] Preferably, the gdhA and leuE genes are overexpressed compared to the unmodified microorganism.

[0102] Preferably, compared to an unmodified microorganism, the microorganism further comprises:

[0103] a) attenuation of the expression of at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda, and gnd, and / or

[0104] b) overexpression of at least one gene selected from the group consisting of pntAB, gdhA, leuE and ygaZH.

[0105] According to a particularly preferred embodiment, the microorganism further comprises attenuation of the udhA, aceEF, sucAB and poxB genes and overexpression of the pta, gdhA and leuE genes.

[0106] In another aspect, a microorganism genetically modified for the production of leucine or isoleucine as described herein is further modified to be able to use sucrose as a carbon source. Preferably, proteins involved in sucrose import and metabolism are overproduced. Preferably, the following proteins are overproduced:

[0107] - CscB sucrose permease, CscA sucrose hydrolase, CscK fructokinase and CscR csc-specific repressor, or

[0108] - ScrA, enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, and the ScrK gene encodes ATP-dependent fructokinase, the ScrB sucrose 6-phosphate hydrolase (invertase), the ScrY sucrose porin, the ScrR sucrose operon repressor.

[0109] Preferably, according to one of the methods provided herein, the gene encoding the protein is overexpressed. Preferably, the microorganism overexpresses:

[0110] - the heterologous cscBKAR gene of Escherichia coli EC3132, or

[0111] - Heterologous scrKYABR gene from Salmonella sp.

[0112] Unless otherwise specified, the names of corresponding genes in Escherichia coli are used to identify genes and proteins (for example, Escherichia coli K12 MG1655 with Genbank accession number U00096.3) in this article. However, in some cases, the use of these names has a wider meaning according to the present invention, and encompasses all corresponding genes and proteins in microorganisms. This is especially true for non-existent (that is, heterologous) genes and proteins (such as GapN, CimA*, etc.) in microorganisms as described herein. The mentioning of any protein (for example, enzyme) or gene provided herein further includes its functional fragment, mutant and functional variant. As provided herein, the functional fragment, mutant and functional variant preferably have at least 90% similarity with the protein or gene, or alternatively, have at least 80%, 90%, 95% or even 100% identity with the protein or gene.

[0113] The degree of sequence identity between proteins is a function of the number of identical amino acid residues or nucleotides at the positions shared by the sequences of the proteins. The term "sequence identity" or "identity" as used herein in the context of two nucleotide or amino acid sequences more specifically relates to residues that are identical in the two sequences when aligned to obtain maximum correspondence. When using a percentage of sequence identity with respect to an amino acid sequence, it should be recognized that the positions where the amino acids are not identical are usually different due to conservative amino acid substitutions, in which the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity). When the sequences differ due to conservative substitutions, the percentage of identity between the sequences can be adjusted upward to correct for the conservativeness of the substitutions. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity". Therefore, the degree of sequence similarity between polypeptides is a function of the number of similar amino acid residues at the positions shared by the sequences of the proteins. The means for identifying similar sequences and their percentages of similarity or their percentages of identity are well known to those skilled in the art, and particularly include BLAST programs, which can be used from the website http: / / www.ncbi.nlm.nih.gov / BLAST / , and their default parameters are indicated on the website. The sequences obtained can then be developed (eg 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) whose default parameters are indicated on these websites.

[0114] Using the reference of known genes given in GenBank, those skilled in the art can determine equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences, which can be determined by sequence alignment 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.

[0115] Specifically, sequence similarity and 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 a similar amino acid or by the same amino acid, then the sequences are similar or identical at that position, respectively.

[0116] Sequence similarity can be expressed as a given amino acid sequence and another amino acid sequence similarity percentage in particular. This refers to the similarity between sequences based on the "similarity score" obtained using a specific amino acid substitution matrix. Such matrices and their use in quantifying the similarity between two sequences are well known in the art and are described in, for example, Dayhoff et al., 1978 and Henikoff and Henikoff, 1992. Sequence similarity can be calculated from the comparison of two sequences, and based on the substitution score matrix and the gap penalty function. As a non-limiting example, the similarity score is determined using the following: BLOSUM62 matrix, gap existence penalty 10 and gap extension penalty 0.1, or BLOSUM62 matrix, gap existence penalty 11 and gap extension penalty 1. Preferably, when using a network-based program (such as BLAST) to determine sequence similarity, no composition adjustment is performed to compensate for the amino acid composition of the compared sequence, and no filter or mask (for example, in order to mask the segment of the sequence with low composition complexity) is applied. The maximum similarity score obtainable for a given amino acid sequence is the similarity score obtained when comparing the sequence to itself. A skilled person is able to determine such a maximum similarity score based on the above parameters for any amino acid sequence. Statistically relevant similarities can also be indicated by a "bit score", as described, for example, in Durbin et al., Biological Sequence Analysis, Cambridge University Press (1998).

[0117] To determine whether a given amino acid sequence has at least 80% similarity to a protein provided herein, the amino acid sequence can be optimally aligned as described above, preferably using the BLOSUM62 matrix, a gap existence penalty of 10, and a gap extension penalty of 0.1. When two sequences are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), a gap existence penalty, and a gap extension penalty to obtain the highest score that the sequence pair may obtain, the sequence is "optimally aligned". For any amino acid sequence, a skilled person can determine 80% similarity, and the maximum score is determined based on the above parameters.

[0118] The similarity percentage or identity percentage as referred to herein is determined after the optimal comparison of the sequence to be compared, which may therefore include one or more insertions, deletions, truncations and / or substitutions. The identity percentage can be calculated by any sequence analysis method well known to those skilled in the art. The similarity percentage or identity percentage can be determined after the sequence to be compared of the sequence taken as a whole is globally compared over its total length. In addition to manual comparison, the algorithm of Needleman and Wunsch (1970) can also be used to determine the global comparison. The optimal comparison of the sequence can preferably be carried out by the global comparison algorithm of Needleman and Wunsch (1970), by the computerized implementation of the algorithm (such as CLUSTAL W) or by visual inspection.

[0119] For nucleotide sequences, sequence comparisons can be performed using any software well known to those skilled in the art (e.g., Needle software). The parameters used can be, among others, the following: "Gap Open" equals 10.0, "Gap Extension" equals 0.5, and an EDNAFULL matrix (NCBI EMBOSS version NUC4.4).

[0120] For amino acid sequences, sequence comparisons can be performed using any software well known to those skilled in the art (e.g., Needle software). The parameters used can be, among others, as follows: "Gap opening" equals 10, "Gap extension" equals 0.1, and the BLOSUM62 matrix.

[0121] Preferably, the percentage similarity or identity as defined herein is determined via a global alignment of the sequences compared over their entire length.

[0122] As a specific example, in order to determine the similarity or identity percentage between two amino acid sequences, the sequences are aligned for optimal comparison. For example, a room can be introduced in the sequence of the first amino acid sequence for optimal comparison with the second amino acid sequence. The amino acid residues at the corresponding amino acid positions are then compared. When the position in the first sequence is occupied by different but conservative amino acid residues, the molecules are similar at the position, and a specific score (for example, as provided in the given amino acid substitution matrix discussed previously) is given. When the 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 the position.

[0123] 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.

[0124] In other words, the percent sequence identity is calculated by comparing the two optimally aligned sequences, determining the number of positions where the same amino acid is present in the two 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.

[0125] PFAM (Protein Families Database of Alignments and Hidden Markov Models; http: / / www.sanger.ac.uk / Software / Pfam / ) represents a large collection of protein sequence alignments that can also be consulted by the skilled artisan. Each PFAM can visualize multiple alignments, view protein domains, assess distribution between organisms, gain access to other databases, and visualize known protein structures.

[0126] Finally, COGs (Clusters of Orthologous Proteins; http: / / www.ncbi.nlm.nih.gov / COG / ) can be obtained by comparing protein sequences from 43 completely sequenced genomes representing 30 major phylogenetic lineages. Each COG is defined by at least three lineages, which allows the identification of previously conserved domains.

[0127] The above definitions and preferred embodiments related to functional fragments and functional variants of proteins apply mutatis mutandis to nucleotide sequences, such as genes, encoding the proteins.

[0128] According to another aspect, the present invention relates to a method for producing leucine and / or isoleucine using the microorganisms described herein. The method comprises the following steps:

[0129] a) culturing a microorganism genetically modified for the production of leucine and / or isoleucine as provided herein in a suitable medium comprising a carbon source, and

[0130] b) recovering leucine and / or isoleucine from the culture medium.

[0131] According to the present invention, the terms "fermentation process", "fermentation production", "fermentation" or "culturing" are used interchangeably to refer to the growth of microorganisms. Such growth is usually carried out in a fermenter with an appropriate growth medium suitable for the microorganism used.

[0132] "Suitable medium" or "medium" refers to a medium optimized for the growth of microorganisms and the synthesis of leucine or isoleucine by cells. A medium (e.g., a sterile liquid medium) contains nutrients necessary or beneficial to the maintenance and / or growth of microorganisms, such as a carbon source or carbon substrate, a nitrogen source; 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. The fermentation process is usually carried out in a reactor with a synthetic (especially inorganic) medium of known determined composition, which is adapted to microorganisms (e.g., Escherichia coli). In particular, the inorganic medium may have a composition identical or similar to that of M9 medium (Anderson, 1946), M63 medium (Miller, 1992), or a medium as defined by Schaefer et al. (1999). "Synthetic medium" refers to a medium containing a chemically determined composition on which an organism grows.

[0133] According to the present invention, the term "carbon source (source of carbon)", "carbon source (carbon source)" or "carbon substrate" refers to any carbon source that can be metabolized by microorganisms, wherein the substrate contains at least one carbon atom. According to the present invention, the carbon source is preferably at least one carbohydrate, and is a mixture of at least two carbohydrates in some cases. The term "carbohydrate" refers to any carbon source that can be metabolized by microorganisms and contains at least three carbon atoms, two hydrogen atoms. One or more carbohydrates can be selected from: monosaccharides (such as glucose, fructose, mannose, galactose, etc.), disaccharides (such as sucrose, cellobiose, maltose, lactose, etc.), oligosaccharides (such as raffinose, stachyose, maltodextrin, etc.), polysaccharides (such as cellulose, starch) or glycerol. Preferred carbon sources are fructose, galactose, glucose, lactose, maltose, sucrose or any combination thereof, more preferably glucose, fructose, galactose, lactose and / or sucrose, most preferably glucose.

[0134] The culture medium preferably contains a nitrogen source that can be used by the microorganism. The nitrogen source can be inorganic (e.g., (NH4)2SO4) or organic (e.g., urea or glutamate). Preferably, the nitrogen source is in the form of ammonium or ammonia. Preferably, the nitrogen source is an ammonium salt, such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium hydroxide and ammonium phosphate; or ammonia gas, corn steep liquor, peptone (e.g., Bacto TMIn some cases, the nitrogen source can be derived from renewable biomass in the following source: microbial source (such as brewer's yeast autolysate, waste yeast autolysate, baker's yeast, hydrolyzed waste cells, algae biomass), plant source (such as cottonseed meal, soy peptone, soy peptide, soy flour (soy flour), soy flour (soybean flour), soy molasses, rapeseed meal, peanut meal, wheat bran hydrolysate, rice bran and defatted rice bran, malt, red lentil flour, black bean, chickpea, mung bean, bean flour, pigeon pea flour, potato processing waste (protamylasse)) or animal source (such as fish waste hydrolysate, fish protein hydrolysate, chicken feather; Feather hydrolysate, meat and bone meal, silkworm larvae, fibroin powder, shrimp waste, beef extract), or any other nitrogenous waste. More preferably, the nitrogen source is peptone and / or yeast extract.

[0135] According to a particularly preferred embodiment, the culture medium comprises at least one carbohydrate, such as glucose, and acetate and / or yeast extract and / or peptone, more preferably glucose and acetate.

[0136] Those skilled in the art are able to define the culture conditions for the microorganism according to the present 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 39°C, even more preferably at about 37°C. In the case of a heat-inducible promoter included in the microorganism provided herein, the microorganism is preferably fermented at about 39°C.

[0137] 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).

[0138] "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.

[0139] Microaerobic conditions are defined as culture conditions in which a low percentage of oxygen (eg, using a gas mixture containing between 0.1% and 15% oxygen, made up to 100% with an inert gas such as nitrogen, helium or argon etc.) is dissolved into the liquid phase.

[0140] 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.

[0141] The term "recovery" as used herein refers to the process of separating or isolating the produced leucine or isoleucine using conventional laboratory techniques known to those skilled in the art. Preferably, step b) of the method comprises a filtration, ion exchange, crystallization and / or distillation step, more preferably a crystallization step. Leucine or isoleucine can be recovered from the culture medium and / or from the microorganism itself. Preferably, at least leucine or isoleucine is recovered from the culture medium.

[0142] Example

[0143] 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.

[0144] method

[0145] The scheme used in the following examples is:

[0146] The present invention uses Solution 1 (Chromosome modification by homologous recombination, selection of recombinants and excision of an 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).

[0147] Scenario 3: Construction of recombinant plasmids.

[0148] Recombinant DNA technology is fully described and known to those skilled in the art. In brief, oligonucleotides (those skilled in the art can define) are used to carry out PCR amplification of DNA fragmentation, and Escherichia coli MG1655 genomic DNA or suitable synthetically synthesized fragments are used as substrates. DNA fragmentation and selected plasmids are digested with compatible restriction enzymes (those skilled in the art can define), then connected and transformed into competent cells. Transformants are analyzed, and the recombinant plasmid of interest is verified by DNA sequencing.

[0149] Solution 4: Evaluation of L-leucine and L-isoleucine fermentation performance.

[0150] The production strains were evaluated using the medium MM_LEF5 (Table 1) for leucine fermentation or the MM_ILF3 (Table 2) for isoleucine fermentation adjusted to pH 6.8 in 500 mL Erlenmeyer baffled flasks. At 30°C, 5 mL pre-cultures were grown for 16 hours in a rich medium (LB medium (10 g / L bactopeptone, 5 g / L yeast extract, 5 g / L NaCl) supplemented with 10 g / L glycerol, 1 g / L acetic acid, 1.5 g / L glutamate and 6 g / L succinate). It was used to inoculate 50 mL of culture to an OD of 0.2. 600 If necessary, antibiotics were added to the culture medium (the final concentration of spectinomycin was 50 mg.L -1 ). The temperature of the culture was 37° C. When the culture reached an absorbance of 2 to 5 uOD / mL at 600 nm, extracellular amino acids were quantified by HPLC after OPA / Fmoc (Agilent Technologies) derivatization, and other relevant metabolites (organic acids and glucose) were analyzed using HPLC with refractive index detection.

[0151] Table 1: Composition of MM LEF5 medium

[0152] Compound <![CDATA[Concentration (g.L -1 )]]> <![CDATA[Citric acid.H2O]]> 1.00 <![CDATA[MgSO4.7H2O]]> 1.00 <![CDATA[CaCl2.2H2O]]> 0.04 <![CDATA[CoCl2.6H2O]]> 0.0080 <![CDATA[MnSO4.H2O]]> 0.0200 <![CDATA[CuCl2.2H2O]]> 0.0020 <![CDATA[H3BO3]]> 0.0010 <![CDATA[Na2MoO4.2H2O]]> 0.0004 <![CDATA[ZnSO4.7H2O]]> 0.0040 <![CDATA[Na2HPO4]]> 2.00 <![CDATA[K2HPO4]]> 2.00 <![CDATA[(NH4)2HPO4]]> 8.00 <![CDATA[(NH4)2SO4]]> 5.00 <![CDATA[NH4Cl]]> 0.13 <![CDATA[FeSO4.7H2O]]> 0.01 Thiamine 0.0116 MOPS 40 glucose 20 Acetic acid 2.5 Glutamate 1.5 Succinic acid 0.6 Yeast Extract 2

[0153] Table 2: Composition of MM ILF3 medium

[0154]

[0155]

[0156] In these cultures, the leucine yield (Y 亮氨酸 ) is expressed as follows:

[0157]

[0158] And the isoleucine yield (Y 异亮氨酸 ) is expressed as follows:

[0159]

[0160] In these cultures, the leucine productivity (P 亮氨酸 ) is expressed as follows:

[0161]

[0162] And the isoleucine productivity (P 异亮氨酸 ) is expressed as follows:

[0163]

[0164] Solution 5: Biomass estimation.

[0165] The spectrophotometer (Nicolet Evolution 100UV-Vis, ) monitors changes in biomass. Biomass production increases the turbidity of the growth medium. It is determined by measuring the absorbance at a wavelength of 600 nm. Each unit of absorbance corresponds to 2.2 x 10 9 + / -2x 10 8 cells / mL.

[0166] Example 1: Strain construction.

[0167] Leucine producing strains: strains 1 to 6.

[0168] Strain 1:

[0169] According to Schemes 1, 2 and 3, strain 1 was obtained by sequentially modifying the E. coli MG1655 strain as follows:

[0170] - by knocking out lactate dehydrogenase (ldhA gene, SEQ ID NO: 1, encoding LdHA of SEQ ID NO: 2), alcohol dehydrogenase (adhE gene, SEQ ID NO: 3, encoding AdhE of SEQ ID NO: 4) and methylglyoxal synthase (mgsA gene, SEQ ID NO: 5 encoding MgsA of SEQ ID NO: 6),

[0171] - by replacing the natural promoter of the small regulatory subunit of acetohydroxyacid synthase I (iilvBN genes: ilvB: SEQ ID NO: 7, encoding IlvB of SEQ ID NO: 8; ilvN: SEQ ID NO: 9, encoding IlvN of SEQ ID NO: 10) (Brosius et al., 1985) with an artificial Ptrc promoter,

[0172] - by overexpressing the following genes in the pCL1920 vector (Lerner & Inouye, 1990) together with the cI857 allele of the thermosensitive repressor of bacteriophage lambda (SEQ ID NO: 13, encoding the thermosensitive repressor protein of SEQ ID NO: 14) (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994), organized into two operons under the control of the PR or PL promoter:

[0173] - ilvE gene, which encodes branched-chain amino acid transaminase (SEQ ID NOs: 15 and 16, respectively),

[0174] - the ilvD gene, which encodes the IlvD dihydroxyacid dehydratase (SEQ ID NOs: 17 and 18, respectively),

[0175] - ilvC gene, encoding an IlvC ketol-acid reductoisomerase (SEQ ID NOs: 19 and 20, respectively),

[0176] - ilvBN gene, which encodes two subunits of acetohydroxyacid synthase I (ilvB: SEQ ID NOs: 7 and 8; ilvN: SEQ ID NOs: 9 and 10),

[0177] The genes are organized into an operon under the control of the PR promoter, and

[0178] -leuA* allele encoding a leucine feedback-resistant (FBR) 2-isopropylmalate synthase with an amino substitution G462D (leuA: SEQ ID NO: 21, encoding LeuA of SEQ ID NO: 22; leuA*: SEQ ID NO: 23, encoding LeuA* of SEQ ID NO: 24)

[0179] -leuB gene, encoding 3-isopropylmalate dehydrogenase (SEQ ID NOs: 25 and 26, respectively), and

[0180] -leuCD gene (leuC: SEQ ID NO: 27 and leuD: SEQ ID NO: 29), encoding 3-isopropylmalate dehydratase subunits (LeuC: SEQ ID NO: 28, and LeuD: SEQ ID NO: 30),

[0181] The genes are organized into an operon under the control of the PL promoter.

[0182] Strain 2:

[0183] According to Schemes 1 and 2, strain 2 was obtained by knocking out citrate synthase (gltA gene, SEQ ID NO: 31, encoding GltA of SEQ ID NO: 32) in strain 1.

[0184] Strain 3:

[0185] According to Schemes 1, 2 and 3, strain 3 was obtained by sequentially modifying strain 1 as follows:

[0186] - by knocking out glyceraldehyde-3-phosphate dehydrogenase A (gapA gene, SEQ ID NO: 33, encoding GapA of SEQ ID NO: 34),

[0187] - by overexpressing the heterologous gapN gene (SEQ ID NO: 35) encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (SEQ ID NO: 36, Uniprot Q59931) by cloning the gene into the pCL1920 vector of strain 1 directly downstream of the PR promoter and upstream of the ilvE gene.

[0188] Strain 4:

[0189] According to Schemes 1, 2 and 3, strain 4 was obtained by sequentially modifying strain 2 as follows:

[0190] - by knocking out glyceraldehyde-3-phosphate dehydrogenase A (gapA gene, SEQ ID NO: 33, encoding GapA of SEQ ID NO: 34),

[0191] - by overexpressing the heterologous gapN gene (SEQ ID NO: 35) encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (SEQ ID NO: 36, Uniprot Q59931) by cloning the gene into the pCL1920 vector of strain 1 directly downstream of the PR promoter and upstream of the ilvE gene.

[0192] Strain 5:

[0193] According to Schemes 1 and 2, strain 5 was obtained by sequentially knocking out the gapB gene encoding D-erythrose-4-phosphate dehydrogenase (SEQ ID NO:45, GapB encoding SEQ ID NO:46) and the gapC pseudogene encoding glyceraldehyde-3-phosphate dehydrogenase (SEQ ID NO:47) (when the gene was intact) in strain 4.

[0194] Strain 6:

[0195] According to Schemes 1, 2 and 3, strain 6 was obtained by sequentially modifying strain 5 as follows:

[0196] - by knocking out soluble pyridine nucleotide transhydrogenase (udhA gene, SEQ ID NO:48, encoding UdhA of SEQ ID NO:49), pyruvate dehydrogenase subunits AceE and AceF (aceEF genes, SEQ ID NOs:50 and 52, encoding AceEF of SEQ ID NOs:51 and 53), pyruvate oxidase (poxB gene, SEQ ID NO:54, encoding PoxB of SEQ ID NO:55) and 2-oxoglutarate dehydrogenase subunits SucA and SucB (sucA gene: SEQ ID NO:56, encoding SucA of SEQ ID NO:57; sucB gene: SEQ ID NO:58, encoding SucB of SEQ ID NO:59),

[0197] - by overproducing phosphate acetyltransferase by adding an artificial Ptrc promoter in front of the pta gene on the chromosome (pta gene, SEQ ID NO: 60, encoding Pta of SEQ ID NO: 61) (Brosius et al., 1985),

[0198] - By overexpressing the gdhA gene encoding glutamate dehydrogenase (gdhA gene, SEQ ID NO:62, encoding GdhA of SEQ ID NO:63) and the leuE gene encoding leucine exporter (leuE gene, SEQ ID NO:64, encoding LeuE of SEQ ID NO:65), this was achieved by cloning the genes into the pCL1920 vector of strain 4 under the control of the PR promoter and the leuE promoter, respectively.

[0199] Isoleucine producing strains: strains 7 to 12.

[0200] Strain 7:

[0201] According to Schemes 1, 2 and 3, strain 7 was obtained by sequentially modifying the E. coli MG1655 strain as follows:

[0202] - Knockout of lactate dehydrogenase (ldhA gene, SEQ ID NO: 1, encoding LdhA of SEQ ID NO: 2), alcohol dehydrogenase (adhE gene, SEQ ID NO: 3, encoding AdhE of SEQ ID NO: 4), methylglyoxal synthase (mgsA gene, SEQ ID NO: 5, encoding MgsA of SEQ ID NO: 6), and 2-isopropylmalate synthase (leuA gene, SEQ ID NO: 21, encoding LeuA of SEQ ID NO: 22)

[0203] - replacement of the acetolactate synthase III small subunit (ilvH gene, SEQ ID NO: 66, encoding IlvH of SEQ ID NO: 67) with the valine and isoleucine FBR ilvH* allele encoding the IlvH* protein with the amino acid substitutions G14D and S17F (Park et al., 2012), and overexpression of the ilvIH* genes (ilvI gene: SEQ ID NO: 72, encoding IlvI of SEQ ID NO: 73, and ilvH* gene of SEQ ID NO: 68, encoding IlvH* of SEQ ID NO: 69) by adding an artificial Ptrc promoter in front of the ilvI gene on the chromosome (Brosius et al., 1985),

[0204] The following genes were overexpressed on the pCL1920 vector (Lerner & Inouye, 1990) together with the cI857 allele of the thermosensitive repressor of bacteriophage lambda (SEQ ID NO: 13, encoding the thermosensitive repressor protein of SEQ ID NO: 14) (amplified from the pFC1 vector, Mermet-Bouvier & Chauvat, 1994), organized into two operons under the control of the PR or PL promoter:

[0205] - ilvE gene, which encodes branched-chain amino acid transaminase (SEQ ID NOs: 15 and 16, respectively),

[0206] - the ilvD gene, which encodes the IlvD dihydroxyacid dehydratase (SEQ ID NOs: 17 and 18, respectively),

[0207] -ilvC gene, encoding IlvC ketol-acid reductoisomerase (SEQ ID NO: 19, respectively).

[0208] and 20),

[0209] - ilvIH gene, which encodes two subunits of acetolactate synthase III (ilvI: SEQ ID NO: 72, encoding IlvI of SEQ ID NO: 73; ilvH: SEQ ID NO: 66, encoding IlvH of SEQ ID NO: 67); more precisely, the ilvH*FBR allele encoding an IlvH protein having the amino acid substitutions G14D and S17F was used (ilvH* gene, SEQ ID NO: 68, encoding IlvH* of SEQ ID NO: 69),

[0210] The genes are organized into an operon under the control of the PR promoter, and

[0211] -cimA* allele (SEQ ID NO:76) encoding an isoleucine feedback resistant (FBR) gene with amino acid substitutions I47V, E114V, H126Q, T204A, L238S, V373STOP

[0212] citramalate synthase CimA* (SEQ ID NO: 77) (cimA gene encoding citramalate synthase CimA originally from Methanococcus jannaschii, SEQ ID NOs: 74 and 75, respectively; Uniprot: Q58787),

[0213] -leuB gene, which encodes 3-isopropylmalate dehydrogenase (SEQ ID NO: 25, respectively).

[0214] and 26), and

[0215] -leuCD gene (leuC: SEQ ID NO: 27 and leuD: SEQ ID NO: 29), encoding 3-isopropylmalate dehydratase subunits (LeuC: SEQ ID NO: 28, and LeuD: SEQ ID NO: 30),

[0216] The genes are organized into an operon under the control of the PL promoter.

[0217] Strain 8:

[0218] According to Schemes 1 and 2, strain 8 was obtained by knocking out citrate synthase (gltA gene, SEQ ID NO: 31, encoding GltA of SEQ ID NO: 32) in strain 7.

[0219] Strain 9:

[0220] According to Schemes 1, 2 and 3, strain 9 was obtained by sequentially modifying strain 7 as follows:

[0221] - by knocking out glyceraldehyde-3-phosphate dehydrogenase A (gapA gene, SEQ ID NO: 33, encoding GapA of SEQ ID NO: 34),

[0222] - by overexpressing the heterologous gapN gene (SEQ ID NO: 35) encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (SEQ ID NO: 36, Uniprot Q59931) by cloning the gene into the pCL1920 vector of strain 7 directly downstream of the PR promoter and upstream of the ilvE gene.

[0223] Strain 10:

[0224] According to Schemes 1, 2 and 3, strain 10 was obtained by sequentially modifying strain 8 as follows:

[0225] - by knocking out glyceraldehyde-3-phosphate dehydrogenase A (gapA gene, SEQ ID NO: 33, encoding GapA of SEQ ID NO: 34),

[0226] - by overexpressing the heterologous gapN gene (SEQ ID NO: 35) encoding the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans (SEQ ID NO: 36, Uniprot Q59931) by cloning the gene into the pCL1920 vector of strain 7 directly downstream of the PR promoter and upstream of the ilvE gene.

[0227] Strain 11:

[0228] According to Schemes 1 and 2, strain 5 was obtained by sequentially knocking out the gapB gene encoding D-erythrose-4-phosphate dehydrogenase (SEQ ID NOs: 46 and 45, respectively) and the gapC pseudogene encoding glyceraldehyde-3-phosphate dehydrogenase (SEQ ID NO: 47) (when the genes were intact) in strain 10 to produce strain 11.

[0229] Strain 12:

[0230] According to Schemes 1, 2 and 3, strain 12 was obtained by sequentially modifying strain 11 as follows:

[0231] - by knocking out soluble pyridine nucleotide transhydrogenase (udhA gene, SEQ ID NO:48, encoding UdhA of SEQ ID NO:49), pyruvate dehydrogenase subunits AceE and AceF (aceEF genes, SEQ ID NOs:50 and 52, encoding AceEF of SEQ ID NOs:51 and 53), pyruvate oxidase (poxB gene, SEQ ID NO:54, encoding PoxB of SEQ ID NO:55) and 2-oxoglutarate dehydrogenase subunits SucA and SucB (sucA gene: SEQ ID NO:56, encoding SucA of SEQ ID NO:57; sucB gene: SEQ ID NO:58, encoding SucB of SEQ ID NO:59),

[0232] - by overproducing phosphate acetyltransferase by adding an artificial Ptrc promoter in front of the pta gene on the chromosome (pta gene, SEQ ID NO: 60, encoding Pta of SEQ ID NO: 61) (Brosius et al., 1985),

[0233] - By overexpressing the gdhA gene encoding glutamate dehydrogenase (SEQ ID NOs: 63 and 62, respectively) and the ygaZH gene encoding the valine exporter (ygaZ gene: SEQ ID NO: 78, encoding YgaZ of SEQ ID NO: 79; ygaH gene: SEQ ID NO: 80, encoding YgaH of SEQ ID NO: 81), this was achieved by cloning the genes into the pCL1920 vector of strain 10 under the control of the PR promoter and the ygaZ promoter, respectively.

[0234] Example 2: Strain performance.

[0235] Leucine production:

[0236] Table 3: Biomass production, leucine titer, productivity and yield of different strains grown on the medium described in Table 1.

[0237]

[0238] The symbol "+" indicates an increase of up to 2 times compared to the value of reference strain 1, the symbol "++" indicates an increase between 2 and 5 times, and "+++" indicates an increase greater than 5 times. The symbol "-" indicates a decrease of up to 2 times compared to the value of reference strain 1, and the symbol "--" indicates a decrease between 2 and 4 times.

[0239] The results obtained using strain 2 (gltA deletion) show the interesting effect of inhibiting carbon flux into the tricarboxylic acid cycle and biomass. The increased amount of carbon is used for leucine production, and the leucine yield is therefore improved.

[0240] As can be seen from Table 3, the coding for reducing NADP + Functional replacement of gapA with the gene for the enzyme (gapN) of the strain leads to improved strain performance. Leucine productivity is particularly increased. However, these modifications affect biomass production. This is clearly observable for strain 3.

[0241] Results obtained using strain 4 (gltA deleted and gapA replaced by gapN) showed that limiting carbon flux into the tricarboxylic acid cycle and using NADPH generating enzymes advantageously improved leucine production, as leucine productivity, titer and yield were increased.

[0242] Similar results were obtained with strains carrying attenuated gltA gene expression rather than a gltA gene deletion in both strain 2 and strain 4 backgrounds (data not shown).

[0243] To ensure that strain 4 was unable to produce NADH via the glycolytic pathway, the gapB and gapC genes were deleted in strain 5. This advantageously resulted in an increase in metabolic stability.

[0244] Strain 6 (deletion of udhA, aceEF, poxB, sucAB, and overexpression of pta, gdhA, leuE) showed further improvement in leucine production, especially in final leucine titer and yield. Advantageously, the gene encoding the NADP-consuming + Inhibition of genes for enzymes of leucine precursors combined with improved acetyl-CoA synthesis increases leucine production. In particular, deletion of the aceEF gene and use of acetate favors leucine production.

[0245] Isoleucine production:

[0246] Table 4: Biomass production, isoleucine titer, productivity and yield of different strains grown on the medium described in Table 2.

[0247]

[0248] The symbol "+" indicates an increase of up to 2 times compared to the value of reference strain 7, the symbol "++" indicates an increase between 2 and 5 times, and "+++" indicates an increase greater than 5 times. The symbol "-" indicates a decrease of up to 2 times compared to the value of reference strain 7, and the symbol "--" indicates a decrease between 2 and 4 times.

[0249] The results obtained using strain 8 (gltA deletion) show the interesting effect of inhibiting carbon flux into the tricarboxylic acid cycle and biomass. The increased carbon amount is used for isoleucine production, and the isoleucine productivity is therefore improved. As can be seen from Table 4, using the encoding reduced NADP + Functional replacement of gapA with the gene for the enzyme (gapN) of the strain 9 resulted in improved strain performance. Isoleucine productivity was particularly increased. However, these modifications affected biomass production. This was clearly observed for strain 9.

[0250] The results obtained using strain 10 (gltA deletion and gapA replaced by gapN) show that limiting the carbon flux into the tricarboxylic acid cycle and using an NADPH generating enzyme advantageously improves isoleucine production. In fact, the productivity, titer and yield of isoleucine were increased.

[0251] Similar results were obtained with strains carrying attenuated gltA gene expression rather than a gltA gene deletion in both strain 8 and strain 10 backgrounds (data not shown).

[0252] To ensure that strain 10 could not produce NADH via the glycolytic pathway, the gapB and gapC genes were deleted in strain 11. This resulted in an increase in metabolic stability.

[0253] Strain 12 (deletion of udhA, aceEF, poxB, sucAB, and overexpression of pta, gdhA, ygaZH) exhibited further improvement in isoleucine production. Advantageously, the final leucine titer and yield were further increased. + Inhibition of genes for enzymes of isoleucine precursors combined with improved acetyl-CoA synthesis increases isoleucine production. In particular, deletion of the aceEF gene and use of acetate favors isoleucine production.

[0254] References

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[0267] Iddar et al.(2005),Int Microbiol.,8(4):251-8.

[0268] Lerner&Inouye,(1990),Nucleic Acids Research,18(15):4631

[0269] Leuchtenberger,et al,(2005)Appl.Microbiol.Biotechnol.69,1-8

[0270] Mermet-Bouvier&Chauvat,(1994),Current Microbiology,28:145-148

[0271] Miller,(1992)“A Short Course in Bacterial Genetics:A LaboratoryManual and Handbook for Escherichia coli and Related Bacteria”,Cold SpringHarbor Laboratory Press,Cold Spring Harbor,N.Y.

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Claims

1. A microorganism genetically modified for the production of leucine and / or isoleucine, wherein the microorganism comprises the following modifications: a) expression of a heterologous gapN gene encoding a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and b) Attenuation of the expression of the gapA and gltA genes compared to an unmodified microorganism.

2. The microorganism according to claim 1, wherein the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase encoded by the gapN gene has at least 80% identity with GapN from Streptococcus mutans. The microorganism according to claim 1 or 2, wherein the gapA gene is deleted.

4. The microorganism according to claim 3, further comprising a reduction in the expression of the gapB and / or gapC genes compared to the unmodified microorganism, preferably a deletion of the gapB and gapC genes.

5. The microorganism according to any one of claims 1 to 4, further comprising overexpression of at least one gene selected from ackA, pta and acs compared to an unmodified microorganism.

6. The microorganism according to any one of claims 1 to 5, wherein the microorganism is genetically modified for the production of leucine and comprises overexpression of the following genes compared to an unmodified microorganism: ilvBN, ilvC, ilvD, leuA*, leuB, leuC, leuD and ilvE.

7. The microorganism according to any one of claims 1 to 5, wherein the microorganism is genetically modified for the production of isoleucine and comprises: a) Expression of heterologous cimA* genes, b) overexpression of the following genes compared to the unmodified microorganism: ilvIH*, ilvC, ilvD, leuB, leuC, leuD and ilvE, and c) Attenuation of the leuA gene.

8. The microorganism according to any one of claims 1 to 7, further comprising, compared to an unmodified microorganism: a) attenuation of the expression of at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda, and gnd, and / or b) overexpression of at least one gene selected from the group consisting of pntAB, gdhA, leuE and ygaZH.

9. The microorganism according to claim 8, wherein at least one gene selected from the group consisting of udhA, aceEF, sucAB, poxB, brnQ, livKHMGF, adhE, ldhA, frdABCD, mgsA, pflAB, zwf, edd, eda, and gnd is deleted.

10. The microorganism according to any one of claims 1 to 9, wherein the microorganism belongs to the genus Escherichia, preferably wherein the microorganism is Escherichia coli; the genus Corynebacterium, preferably wherein the microorganism is Corynebacterium glutamicum; or the genus Streptococcus, preferably wherein the microorganism is selected from Streptococcus thermophilus and Streptococcus salivarius; more preferably wherein the microorganism is Escherichia coli.

11. A method for producing leucine and / or isoleucine, the method comprising the steps of: a) cultivating the microorganism genetically modified for the production of leucine and / or isoleucine according to any one of claims 1 to 10 in a suitable medium comprising a carbon source, and b) recovering leucine and / or isoleucine from the culture medium.

12. The method of claim 11, wherein the culture medium further comprises acetate.

13. The method according to any one of claims 11 or 12, wherein the carbon source is glucose, fructose, galactose, lactose and / or sucrose.

14. The process according to any one of claims 11 to 13, wherein step b) comprises a crystallization step.

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