Recombinant host cells for anthranilic acid production

Abstract

The present invention relates to recombinant bacteria that have been genetically modified to produce anthranilic acid and to be capable of growing in tryptophan-deficient culture media, and to methods for producing anthranilic acid using said recombinant bacteria.

Description

Technical Field

[0001] The present invention relates to the production of anthranilic acid, particularly to the field of production of anthranilic acid using recombinant host cells.

Background Art

[0002] Anthranilic acid is a natural intermediate in the shikimic acid pathway and is a precursor for the biosynthesis of the aromatic amino acid L-tryptophan. It is industrially used as a synthetic intermediate for dyes, fragrances, pharmaceuticals, and other useful products.

[0003] The chemical synthesis of anthranilate requires high temperature and pressure conditions and non-renewable naphthalene, and is a non-sustainable and expensive process that generates toxic by-products. Therefore, there is a strong demand for a sustainable technology that provides a renewable and biologically derived source of anthranilate.

[0004] Some microorganisms and plants have the metabolic ability to synthesize anthranilate. In particular, the biosynthetic pathways of anthranilic acid and aromatic acids in bacteria are fairly well understood. As shown in the figure, FIG. 1 shows a simplified scheme of this pathway in Escherichia coli. Briefly, the first intermediate, 3-deoxy-D-arabino-heptulosonate-7-phosphate, is generated from two precursors, erythrose 4-phosphate (E4P) from the pentose phosphate pathway and phosphoenolpyruvate (PEP) from glycolysis, by AroF, AroH or AroG of DAHP synthase. Then, aroB, aroD, aroE catalyze three reactions from DAHP to produce shikimic acid (SHK). Subsequently, chorismic acid is synthesized from SHK by AroL / K, AroA, AroC. Finally, TrpE and TrpG catalyze the production of anthranilic acid from chorismic acid. Then, anthranilic acid is used for the production of tryptophan through several reactions continuously catalyzed by the enzymes TrpD, TrpC, TrpA, and TrpB. Therefore, as a metabolic intermediate, anthranilic acid does not accumulate and is not secreted.

[0005] Early studies on the L-tryptophan operon of Escherichia coli (E. coli) identified anthranilate-secreting mutants (Yanofsky et al., Genetics, 1971, 69, pp. 409-433). Characterization of one of the strains obtained by UV mutagenesis (W3110 trpD9923) revealed a mutation in the gene encoding the bifunctional protein TrpGD, which connects to a stop codon and a non-functional TrpD domain. In an attempt to provide a microbial strain for anthranilate production, this strain was further modified to overexpress genes encoding feedback-resistant DAHP synthase, transketolase, glucokinase, and galactose permyase (Balderas-Hernandez et al., Microb Cell Fact. April 2, 2009; 8:19). Similarly, anthranilate accumulation has also been confirmed in mutant Bacillus strains (US Patent No. 5,422,256).

[0006] However, a significant problem is that these strains are tryptophan-dependent. When using tryptophan-dependent strains, tryptophan must be added to the culture medium, dramatically increasing costs in industrial production. As a result, there is still a strong need for well-improved methods to achieve industrially adequate anthranilic acid productivity using tryptophan-non-tryptophan-dependent bacterial strains. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 5,422,256 [Non-patent literature]

[0008] [Non-Patent Document 1] Yanofsky et al., Genetics, 1971, 69, pp. 409-433. [Non-Patent Document 2] Balderas-Hernandez, Microb Cell Fact. April 2, 2009; 8:19 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol 48:443 pages [Non-licensed Document 4] Smith and Waterman, Adv. Appl. Math. 2:482, 1981 [Non-licensed Document 5] Altschul, 1997, Nucleic Acids Res. 25: 3389~3402 pages. [Non-licensed Document 6] Altschulら2005, FEBS J. 272:5101~5109 pages [Non-licensed Document 7] http: / / blast.ncbi.nlm.nih.gov / [Non-licensed Document 8] http: / / www.ebi.ac.uk / Tools / emboss / [Non-licensed Document 9] Horowitz and Platt, Journal of Molecular Biology, 156.2 (1982), pages 257~67 [Non-licensed Document 10] Sambrookら, 2012; Molecular cloning: a laboratory manual, 4th edition Cold Spring Harbor [Non-licensed Document 11] Jossek, FEMS Microbiol Lett, August 7, 2001; 202(1): 145-8 pages. [Non-licensed Document 12] MascarenhasらApplied and Environmental Microbiology, October 1991, 57;10:2995~2999 pages [Non-licensed Document 13] Ray et al., Journal of Bacteriology, December 1988, 170(12), pp. 5500-5506. [Non-Patent Document 14] Kirill et al. PNAS, June 2000, 97 (12) pp. 6640-6645 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present inventors aim to produce anthranilic acid in recombinant bacterial host cells that are tryptophan-non-nutrient-requiring strains, i.e., strains that can grow in tryptophan-deficient culture media. [Means for solving the problem]

[0010] Accordingly, in a first aspect, the present invention relates to a method for producing anthranilic acid, comprising the steps of culturing a recombinant bacterium genetically modified to induce an imbalance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity with preference for anthranilate synthase activity, and to be able to grow in tryptophan-deficient culture medium, and optionally recovering anthranilic acid. In a preferred embodiment, the non-modified bacterium corresponding to the recombinant bacterium expresses a bifunctional protein exhibiting TrpG activity and TrpD activity, and the recombinant bacterium is genetically modified to separately express a polypeptide exhibiting glutamine amide transferase activity (TrpG) and a polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD).

[0011] In particular, the present invention relates to a method for producing anthranilic acid, comprising a step of culturing recombinant bacteria and, optionally, a step of recovering anthranilic acid, wherein the unmodified bacteria corresponding to the recombinant bacteria express a bifunctional protein exhibiting TrpG activity and TrpD activity, and the recombinant bacteria are (i) a polypeptide exhibiting glutamine amidotransferase activity (TrpG) and not exhibiting anthranilate phosphoribosyltransferase activity, and (ii) a polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD) and not exhibiting glutamine amidotransferase activity, and are genetically modified so as to individually express them and to reduce the ratio between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity as compared to the unmodified bacteria, and the recombinant bacteria can grow in a tryptophan-deficient culture medium.

[0012] Preferably, the polypeptide exhibiting glutamine amidotransferase activity (TrpG) does not exhibit anthranilate phosphoribosyltransferase activity, and the polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD) does not exhibit glutamine amidotransferase activity.

[0013] The recombinant bacteria may be genetically modified so as to suppress the expression of the TrpD domain of the endogenous bifunctional protein TrpGD, preferably by deleting all or part of the nucleic acid sequence encoding said domain.

[0014] Preferably, the recombinant bacteria are further genetically modified so as to suppress the expression of the endogenous bifunctional protein TrpGD, preferably by deleting all or part of the nucleic acid sequence encoding said protein.

[0015] In particular, the bacterium can be Escherichia coli.

[0016] Recombinant bacteria may include (i) a recombinant nucleic acid comprising a gene encoding glutamine amide transferase (TrpG) or a first operon under the control of a first promoter, wherein the first operon comprises at least a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding anthranilate synthase (TrpE), and / or (ii) a recombinant nucleic acid comprising a gene encoding anthranilate phosphoribosyltransferase (TrpD) or a second operon under the control of a second promoter, wherein the second operon comprises at least a nucleic acid sequence encoding TrpD. In some specific embodiments, the first promoter is more potent than the second promoter. Preferably, the first promoter is a promoter that is not operably linked to the gene encoding TrpG in the unmodified bacteria, and / or the second promoter is a promoter that is not operably linked to the gene encoding TrpD in the unmodified bacteria. The second operon may further include a nucleic acid sequence encoding indole-3-glycerol phosphate synthase enzyme (TrpC), a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase enzyme (TrpF), a nucleic acid sequence encoding the α subunit (TrpA) of tryptophan synthase enzyme, and / or a nucleic acid sequence encoding the β subunit (TrpB) of tryptophan synthase enzyme.

[0017] Alternatively, recombinant bacteria may contain genes encoding glutamine amide transferase (TrpG) and anthranilate phosphoribosyltransferase (TrpD) under the control of the same promoter, thereby enabling the expression of two distinct proteins, TrpG and TrpD.

[0018] Recombinant bacteria may also be genetically modified to express a gene encoding a feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase enzyme, and / or to overexpress an endogenous gene encoding a transketolase enzyme, or to express a heterologous gene encoding a transketolase enzyme.

[0019] Preferably, in the method of the present invention, recombinant bacteria are cultured in a culture medium lacking tryptophan or any tryptophan source.

[0020] In another embodiment, the present invention relates to recombinant bacteria as defined above. In particular, the present invention relates to recombinant Escherichia coli genetically modified to include a nucleic acid sequence encoding glutamine amide transferase (TrpG) under the control of a first promoter and a nucleic acid sequence encoding anthranilate phosphoribosyltransferase (TrpD) under the control of a second promoter. Preferably, the recombinant bacteria include (i) a gene or first operon encoding glutamine amide transferase (TrpG) under the control of a first promoter, wherein the first operon comprises at least a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding anthranilate synthase (TrpE), and (ii) a gene or second operon encoding anthranilate phosphoribosyltransferase (TrpD) under the control of a second promoter. A second operon comprising a gene encoding TrpD or a second operon, wherein the second operon comprises at least a nucleic acid sequence encoding TrpD, and optionally a nucleic acid sequence encoding indole-3-glycerol phosphate synthase enzyme (TrpC), a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase enzyme (TrpF), a nucleic acid sequence encoding the α subunit of tryptophan synthase enzyme (TrpA), and / or a nucleic acid sequence encoding the β subunit of tryptophan synthase enzyme (TrpB).

[0021] The present invention also relates to the use of recombinant bacteria of the present invention for the production of anthranilic acid. [Brief explanation of the drawing]

[0022] [Figure 1] This is a simplified diagram of the biosynthesis of anthranilic acid and aromatic amino acids. [Modes for carrying out the invention]

[0023] This invention relates to recombinant bacteria that are genetically modified to accumulate anthranilic acid while being able to grow in tryptophan-deficient culture media. The invention also relates to the use of such recombinant bacteria for producing anthranilic acid, and to a method for producing anthranilic acid using such recombinant bacteria.

[0024] In this application, the inventors have demonstrated that tryptophan production can be restricted and anthranilates accumulated by altering and controlling the carbon flux to the tryptophan pathway. In fact, the inventors have demonstrated that bacteria, particularly Escherichia coli strains, can be genetically engineered to adjust the ratio between (i) anthranilate phosphoribosyltransferase activity and (ii) anthranilate synthase activity. By reducing the aforementioned ratio, the inventors have demonstrated that engineered strains can produce and accumulate a considerable amount of anthranilic acid while still being able to grow in tryptophan-deficient culture media. In contrast to the anthranilate-producing bacteria described in the prior art, the recombinant bacteria of the present invention do not exhibit tryptophan nutritional requirements. These bacteria do not require any tryptophan supplementation and therefore offer a significant advantage over prior art strains in the industrial production of anthranilic acid.

[0025] definition In the context of this invention, the term "recombinant bacteria" means bacteria that are not found in nature and that contain a modified genome as a result of the deletion, insertion, or alteration of one or more gene elements.

[0026] "Recombinant nucleic acid" or "recombinant nucleic acid molecule" means a nucleic acid (e.g., DNA, cDNA, or RNA molecule) that has been manipulated and is not found in natural or wild-type bacteria. Typically, this term refers to a nucleic acid molecule that contains and / or ligated segments produced using recombinant DNA techniques such as molecular cloning and nucleic acid amplification. Recombinant nucleic acid molecules contain one or more sequences that do not exist in nature and / or ligated nucleic acid molecules that originate from different sources and are not naturally bound together.

[0027] The term "gene" refers to any nucleic acid that codes for a protein. This term encompasses DNA, e.g., cDNA or gDNA, and RNA. Genes can first be prepared, for example, by recombinant techniques, enzymatic techniques, and / or chemical techniques, and then replicated in host cells or in vitro systems. Genes typically contain an open reading frame that codes for a desired protein. Genes may also contain additional sequences, e.g., transcription terminators or signal peptides.

[0028] As used herein, the term "operon" refers to a functional unit of DNA containing two or more genes under the control of a single promoter.

[0029] As used herein, the term “expression cassette” means a nucleic acid construct comprising operably ligated coding regions, i.e., one or more genes, and regulatory regions, i.e., one or more regulatory sequences including a transcription promoter. Optionally, an expression cassette may comprise several coding regions operably ligated to several regulatory regions. In particular, an expression cassette may comprise several coding sequences, each of which is operably ligated to the same promoter or different promoters. Alternatively, an expression cassette may comprise one or more coding sequences, each of which is operably ligated to a separate promoter, and several other coding sequences operably ligated to a common promoter.

[0030] The term "operably coupled" refers to an arrangement in which the control sequence is positioned appropriately relative to the code sequence so that the control sequence directs the expression of the code sequence.

[0031] The term "regulatory sequence" refers to a nucleic acid sequence necessary for gene expression. Regulatory sequences may be native or heterologous. Well-known regulatory sequences currently in use by those skilled in the art are preferred. Such regulatory sequences include, but are not limited to, leaders, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, ribosome binding sites, and transcriptional terminators. Preferably, the regulatory sequence includes a promoter and a transcriptional terminator.

[0032] As used herein, the term “expression vector” means a DNA or RNA molecule containing an expression cassette. Preferably, the expression vector is a linear or circular double-stranded DNA molecule.

[0033] As used herein, the terms “native” or “endogenous” refer to a gene element or protein that is naturally present in a host cell. The term “heterogeneous” refers to a gene element or protein that is not naturally present in a host cell. Preferably, this term refers to a gene element or protein provided from a cell of a different species or genus than the host cell.

[0034] As used herein, the terms “sequence identity” or “identity” refer to the number (%) of identical amino acid residues in the alignment of two polypeptide sequences. Sequence identity is determined by comparing sequences that have been aligned to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using one of several mathematical global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm that optimally aligns the sequences over their entire length (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970, J. Mol. Biol 48:443), while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, Adv. Appl. Math. 2:482, 1981) or Altschul algorithm (Altschul et al. 1997, Nucleic Acids Res. 25:3389-3402; Altschul et al. 2005, FEBS J. 272:5101-5109)). Alignment for determining the percentage of amino acid sequence identity can be achieved by various methods within the scope of the art, for example, by using publicly available computer software available on the internet (e.g., http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve the greatest possible alignment over the entire length of the sequences being compared.Preferably, for the purposes of this specification, the amino acid sequence identity value % refers to a value generated using the pairwise sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to create the optimal global alignment of two sequences, where all search parameters are set to their default values, i.e., scoring matrix=BLOSUM62, Gap open=10, Gap extend=0.5, End gap penalty=false, End gap open=10, and End gap extend=0.5. In some specific embodiments, all sequence identities referred to herein are identical and are set to at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some more specific embodiments, all sequence identities referred to herein are identical and are set to at least 80% sequence identity. In some other specific embodiments, all sequence identities referred to herein are identical and set to at least 90% sequence identities.

[0035] The terms "peptide," "oligopeptide," "polypeptide," and "protein" are interchangeable and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain.

[0036] As used herein, the term “activity” of an enzyme or enzyme complex refers to its function, and in the context of the present invention, it means the reaction catalyzed by the enzyme or complex. Enzyme activity can be measured by any method known to those skilled in the art.

[0037] As used herein, the terms “overexpression” and “increased expression” are interchangeable and mean that the expression of a gene or enzyme is increased compared to an unmodified bacterium. Increased enzyme expression is usually obtained by increasing the expression of the gene encoding the enzyme. In embodiments where the gene or enzyme is not naturally present in the bacteria of the present invention, the terms “overexpression” and “expression” can be used interchangeably. To increase gene expression, those skilled in the art can use any known technique, such as increasing the copy number of the gene in the bacterium, using a promoter that induces a high level of gene expression, i.e., a strong promoter, using an element that stabilizes the corresponding messenger RNA, or modifying the ribosome-binding site (RBS) sequence and the surrounding sequence. In particular, overexpression can be obtained by increasing the copy number of the gene in the bacterium. One or more copies of a gene can be introduced into the genome by recombination methods known to those skilled in the art, including gene substitution or multicopy insertion. Preferably, the expression cassette containing the gene is incorporated into the genome, preferably under the control of a strong promoter. Alternatively, the gene may be carried by an expression vector, preferably a plasmid, which includes an expression cassette in which the gene of interest is preferably placed under the control of a strong promoter. Depending on the nature of the origin of replication, the expression vector may be present in one or more copies within the bacterium. Overexpression of the gene can also be obtained by using a promoter that induces a high level of gene expression. For example, the promoter of an endogenous gene can be replaced with a stronger promoter, i.e., a promoter that induces a higher level of expression. Promoters suitable for use in the present invention are known to those skilled in the art and may be constitutive or inductive, and are preferably constitutive.

[0038] As used herein, the term “reduced expression” means that the expression of a gene or enzyme is reduced compared to that of an unmodified bacterium. Reduced enzyme expression is usually obtained by reducing the expression of the gene encoding the enzyme. To reduce gene expression, those skilled in the art can use any known technique, such as reducing the copy number of the gene in the bacterium and / or using a promoter that induces a lower level of gene expression, i.e., a weak promoter. Preferably, reduced gene expression is obtained by using a promoter that induces a lower level of gene expression. For example, the promoter of an endogenous gene can be replaced with a weak promoter, i.e., a promoter that induces a lower level of expression. Promoters suitable for use in the present invention are known to those skilled in the art and may be constitutive or inductive, preferably constitutive.

[0039] As used herein, the term “unmodified bacteria” refers to wild-type bacteria (i.e., bacteria of natural origin) or corresponding bacteria that have not been genetically modified to accumulate anthranilates, particularly those that have not been genetically modified to modulate the expression levels of genes encoding anthranilate phosphoribosyltransferase (TrpD), glutamine amide transferase (TrpG), and / or anthranilate synthase (TrpE), and / or to modulate anthranilate phosphoribosyltransferase activity and / or anthranilate synthase activity. In particular, compared to wild-type bacteria, corresponding bacteria may include genetic modifications that provide advantages for the industrial production of anthranilates, such as enzymatic activity for antibiotic resistance or broader substrate range, although these modifications are not directly related to anthranilate production. In preferred embodiments, the term refers to wild-type bacteria (i.e., bacteria of natural origin).

[0040] The terms "antranylate phosphoribosyltransferase" or "TrpD" refer to polypeptides exhibiting anthranylate phosphoribosyltransferase activity (EC: 2.4.2.18), i.e., peptides that catalyze the transfer of the phosphoribosyl group of 5-phosphorylribose-1-pyrophosphate to anthranylate, thereby forming N-(5'-phosphoribosyl)-antranylate. Anthranylate phosphoribosyltransferase activity can be determined by any method known to those skilled in the art. For example, the activity can be evaluated by fluorescence analysis at 25°C by measuring the consumption rate of the anthranylate (excitation wavelength, 310 nm; emission, 400 nm) using a spectrofluorometer.

[0041] The terms "glutamine amide transferase" or "TrpG" refer to a polypeptide exhibiting glutamine amide transferase activity, i.e., a polypeptide that produces ammonia as a substrate, which is used in a second step with chorismate and catalyzed by TrpE to produce anthranilate. The glutamine amide transferase activity can be determined by any method known to those skilled in the art. For example, the activity can be evaluated by monitoring the formation of p-nitroanilide from ly-glutamyl-p-nitroanilide at 384 nm using a spectrophotometer.

[0042] The terms “anthranylate synthase” or “TrpE” refer to polypeptides exhibiting anthranylate synthase activity (EC: 4.1.3.27), i.e., polypeptides that catalyze the conversion of colismate and glutamine to anthranylate, glutamate, and pirubate. In Escherichia coli, this polypeptide is encoded by the trpE gene. As used herein, “anthranylate synthase activity” refers to the activity that converts colismate and glutamate to anthranylate, glutamate, and pirubate, i.e., the activity resulting from a combination of TrpE activity and TrpG activity. Anthranylate synthase activity can be determined by any method known to those skilled in the art. For example, the activity can be evaluated by monitoring anthranylate formation from colismate by tracking the increase in fluorescence (excitation wavelength, 310 nm; emission, 400 nm) with a spectrofluorometer.

[0043] The term "indole-3-glycerol phosphate synthase" or "TrpC" refers to a polypeptide exhibiting indole-3-glycerol phosphate synthase activity (EC: 4.1.1.48). The term "phosphoribosyl anthranylate isomerase" or "TrpF" refers to a polypeptide exhibiting phosphoribosyl anthranylate isomerase activity (EC: 5.3.1.24). In a wide variety of bacteria, including Escherichia coli, TrpC and TrpF are part of a bifunctional enzyme that catalyzes two consecutive steps in the tryptophan biosynthesis pathway. The first reaction is catalyzed by an isomerase encoded by the TrpF domain. The second reaction is catalyzed by a synthase encoded by the TrpC domain.

[0044] The term "alpha subunit of tryptophan synthase" or "TrpA" refers to the alpha subunit of tryptophan synthase, i.e., the polypeptide that catalyzes the conversion of 1-C-(indole-3-yl)glycerol 3-phosphate to indole and D-glyceraldehyde 3-phosphate (EC: 4.2.1.20). The term "beta subunit of tryptophan synthase" or "TrpB" refers to the beta subunit of tryptophan synthase. The indole formed by the alpha subunit moves to the beta subunit, where, in the presence of pyridoxal 5'-phosphate, it combines with L-serine to form L-tryptophan.

[0045] The nomenclature for the enzymes and coding genes identified above may differ depending on the organism. However, for simplicity, these terms are used herein independently of the origin of the enzyme or gene.

[0046] As used herein, the term “tryptophan-dependent” refers to bacteria that are unable to synthesize the tryptophan required for their growth. Such bacteria cannot grow in culture media lacking tryptophan or any tryptophan source. Conversely, the term “tryptophan-non-dependent” refers to bacteria that are able to synthesize the tryptophan required for their growth. Such bacteria can grow in culture media lacking tryptophan or any tryptophan source.

[0047] In this application, the terms “anthranilic acid,” “o-aminobenzoic acid,” “2-aminobenzoic acid,” and “anthranilate” are interchangeable and refer to an aromatic acid having the following formula (I) (CAS number: 118-92-3).

[0048] [ka]

[0049] The inventors have discovered that bacteria can be genetically engineered to accumulate anthranilates by controlling the carbon flux to the tryptophan pathway. In fact, the inventors have demonstrated that recombinant bacteria genetically modified to induce an imbalance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity, prioritizing anthranilate synthase activity, can grow in tryptophan-free culture media while accumulating anthranilic acid.

[0050] Accordingly, in a first aspect, the present invention relates to recombinant bacteria genetically modified to induce an imbalance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity, with preference for anthranilate synthase activity. The present invention also relates to recombinant bacteria genetically modified to reduce the ratio between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity compared to unmodified bacteria.

[0051] The recombinant bacteria of the present invention still exhibit anthranilate phosphoribosyltransferase activity and can therefore grow in tryptophan-deficient culture media, i.e., they are not tryptophan-dependent. In other words, in the bacteria of the present invention, the ratio between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity is greater than zero. In fact, the TrpD activity remains sufficient to enable tryptophan synthesis and therefore allows bacterial growth in culture media lacking tryptophan or any tryptophan source.

[0052] Anthranilates are intermediates in the tryptophan biosynthesis pathway and do not accumulate in wild-type bacteria that do not exhibit tryptophan nutritional requirements. The recombinant bacteria of this invention include genetic modifications designed to alter the carbon flux through the tryptophan pathway. These genetic modifications aim to induce an imbalance between anthranylate synthase activity and TrpD activity, prioritizing anthranylate synthase activity. Thus, the recombinant bacteria produce more anthranylates, i.e., as substrates for TrpD, than is required / used in tryptophan biosynthesis. This imbalance leads to the accumulation and secretion of anthranylates, but tryptophan is still produced by the bacteria due to the residual activity of TrpD.

[0053] Anthranilate synthase activity is due to a multimeric complex that catalyzes the two-step biosynthesis of anthranilates. In the first step, TrpG provides glutamine amide transferase activity that produces ammonia as a substrate. This substrate is used in the second step together with colismate and catalyzed by TrpE to produce anthranilates. Therefore, anthranilate synthase activity is a result of TrpG and TrpE activity. In E. coli, anthranilate synthase is encoded by the trpE gene, and the amino-terminal region of the bifunctional protein encoded by the trpGD gene is responsible for glutamine amide transferase (TrpG) activity. In fact, in a wide variety of bacteria, including E. coli, bifunctional proteins are responsible for both TrpG and TrpD activity. For example, in Escherichia coli, the amino-terminal region of the bifunctional protein encoded by the trpGD gene is responsible for TrpG activity, while the carboxy-terminal region of this protein is responsible for TrpD activity. Through this invention, the inventors have found that by separating the expression of the TrpG domain of this bifunctional protein from the expression of the TrpD domain, it is possible to fine-tune the balance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity.

[0054] In the recombinant bacteria of the present invention, the imbalance between anthranylate phosphoribosyltransferase activity and anthranylate synthase activity can be achieved in various different ways, but anthranylate synthase activity is always preferred. Despite this imbalance, the recombinant bacteria still exhibit sufficient levels of anthranylate phosphoribosyltransferase activity to synthesize the tryptophan required for their growth. In some embodiments, TrpE activity and / or TrpG activity are increased. In these embodiments, TrpD activity may remain unchanged, decrease, or increase, but to a smaller extent than the anthranylate synthase activity resulting from TrpE and TrpG activity. Preferably, in these embodiments, TrpD activity remains unchanged or decreases. In some preferred embodiments, TrpD activity is decreased. In these embodiments, anthranylate synthase activity resulting from TrpE and TrpG activity may remain unchanged, increase, or decrease, but to a smaller extent than the TrpD activity. Preferably, in these embodiments, the anthranilate synthase activity resulting from TrpE activity and TrpG activity remains unchanged or increases.

[0055] As used herein, the term “increased activity” refers to increased enzyme activity in recombinant bacteria of the present invention compared to unmodified bacteria. This increase may be due to increased specific catalytic activity, increased specificity to substrates, increased stability of proteins or RNA, and / or increased intracellular concentration of the enzyme or enzyme complex responsible for this activity. Preferably, the increase in activity is due to increased intracellular concentration of the enzyme obtained by overexpression of the gene encoding the enzyme (overexpression of an endogenous gene or expression of a heterologous gene). If the enzyme activity is due to the activity of two distinct polypeptides (e.g., TrpE and TrpG for anthranilate synthase activity), the increase in activity may be due to increased intracellular concentration of one or both, preferably both, of these polypeptides obtained by overexpressing the gene encoding the polypeptides.

[0056] As used herein, the term “reduced activity” refers to reduced enzyme activity in recombinant bacteria of the present invention compared to unmodified bacteria. This reduction may be due to a decrease in specific catalytic activity, a decrease in specificity to a substrate, a decrease in the stability of a protein or RNA, and / or a decrease in the intracellular concentration of the enzyme or enzyme complex responsible for this activity. In some embodiments, the reduction in activity may be due to a decrease in the intracellular concentration of the enzyme. This decrease in intracellular concentration of the enzyme can be achieved by reducing the expression level of the gene encoding the enzyme, or by changes that affect RNA stability and therefore translation efficiency. If the enzyme activity is due to the activity of two distinct polypeptides (e.g., TrpE and TrpG for anthranilate synthase activity), the reduction in activity may be due to a decrease in the intracellular concentration of one or both of these polypeptides. In some other embodiments, the reduction in activity may be due to a decrease in the specific activity of the enzyme. In particular, specific activity may be reduced by structural changes, for example, when the domains of a bifunctional protein are expressed separately from the other domain. As used herein, this term excludes the complete suppression of the enzyme activity.

[0057] As described above, the inventors have found that by cleaving the expression of the TrpG polypeptide from the expression of the TrpD polypeptide, it is possible to fine-tune the balance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity. Therefore, in a more specific embodiment, the present invention relates to recombinant bacteria genetically modified to cleave the expression of the TrpG polypeptide from the expression of the TrpD polypeptide.

[0058] Recombinant bacteria contain a nucleic acid sequence encoding glutamine amide transferase (TrpG) and a nucleic acid sequence encoding anthranylate phosphoribosyltransferase (TrpD), resulting in the expression of two distinct polypeptides: one exhibiting glutamine amide transferase activity (TrpG; not exhibiting anthranylate phosphoribosyltransferase activity) and another exhibiting anthranylate phosphoribosyltransferase activity (TrpD; not exhibiting glutamine amide transferase activity). The expression of the nucleic acid sequences encoding glutamine amide transferase (TrpG) and anthranylate phosphoribosyltransferase (TrpD) may be under the control of the same promoter or two different promoters. Preferably, recombinant bacteria contain / express a nucleic acid sequence encoding glutamine amide transferase (TrpG) under the control of a first promoter and a nucleic acid sequence encoding anthranylate phosphoribosyltransferase (TrpD) under the control of a second promoter.

[0059] As used herein, the terms “nucleotide sequence encoding TrpG,” “nucleotide sequence encoding glutamine amide transferase,” or “gene encoding TrpG” refer to nucleic acids encoding polypeptides that exhibit glutamine amide transferase activity but not anthranilate phosphoribosyl transferase activity, such as polypeptides corresponding only to the TrpG domain of the bifunctional protein TrpGD. Similarly, the terms “nucleotide sequence encoding TrpD,” “nucleotide sequence encoding anthranilate phosphoribosyl transferase,” or “gene encoding TrpD” refer to nucleic acids encoding polypeptides that exhibit anthranilate phosphoribosyl transferase activity but not glutamine amide transferase activity, such as polypeptides corresponding only to the TrpD domain of the bifunctional protein TrpGD.

[0060] Preferably, in bacteria in their wild-type form in which endogenous TrpG activity and TrpD activity are expressed as the bifunctional protein TrpGD, the recombinant bacteria of the present invention are genetically modified to suppress the expression of the endogenous bifunctional protein TrpGD. The endogenous TrpGD gene can be inactivated by any method known to those skilled in the art, for example, by deleting all or part of the gene, by introducing a mutation that induces a nonsense codon or frameshift, or by inserting a gene or expression cassette. In certain embodiments, the expression of only one of the domains of the endogenous bifunctional protein TrpGD, i.e., TrpD or TrpG, is suppressed, and more preferably, the expression of only the TrpD domain is suppressed. The expression of the TrpD domain can be suppressed by any method known to those skilled in the art, for example, by deleting all or part of the nucleic acid sequence encoding the domain, by introducing a mutation that induces a nonsense codon or frameshift, or by inserting a gene or expression cassette. Preferably, TrpD domain expression is suppressed by deleting all or part of the nucleic acid sequence encoding the domain. In some embodiments, particularly when the bacterium is Escherichia coli, TrpD domain expression is suppressed by deleting part of the nucleic acid sequence encoding the domain while maintaining the activity of the internal promoter TrpCp, which is an internally low-efficiency promoter not suppressed by tryptophan (Horowitz and Platt, Journal of Molecular Biology, 156.2 (1982), pp. 257-257).

[0061] In embodiments in which the endogenous nucleic acid sequence encoding TrpD activity is inactivated, the recombinant bacteria may be further modified by introducing an expression cassette containing an endogenous gene or heterologous gene encoding TrpD. In these embodiments, the expression of the endogenous TrpG polypeptide is preferably maintained.

[0062] In embodiments in which endogenous genes encoding TrpD activity and TrpG activity are inactivated, the recombinant bacteria are further modified by introducing one or more expression cassettes containing an endogenous or heterologous gene encoding TrpG and an endogenous or heterologous gene encoding TrpD, resulting in the expression of two distinct polypeptides. In particular, the recombinant bacteria may be further modified by introducing an expression cassette containing an endogenous or heterologous gene encoding TrpG and an expression cassette containing an endogenous or heterologous gene encoding TrpD.

[0063] In bacteria in which TrpG and TrpD activity are naturally expressed as the bifunctional protein TrpGD, the terms "endogenous gene encoding TrpG" and "endogenous gene encoding TrpD" refer to the endogenous nucleic acid sequence encoding the bifunctional protein domain TrpG and the endogenous nucleic acid sequence encoding the bifunctional protein domain TrpD, respectively. The TrpD domain or polypeptide does not exhibit glutamine amide transferase activity, and the TrpG domain or polypeptide does not exhibit anthranilate phosphoribosyltransferase activity.

[0064] As described above, the TrpG polypeptide and TrpD polypeptide expressed in the recombinant bacteria of the present invention may be endogenous polypeptides or heterologous polypeptides (both may be endogenous or heterologous, or one may be endogenous and the other heterologous). In particular, the TrpG polypeptide and TrpD polypeptide may be any known TrpG polypeptide and TrpD polypeptide, preferably selected from bacterial TrpG polypeptide and TrpD polypeptide. In particular, the TrpG polypeptide and TrpD polypeptide may be selected from the TrpG domain and TrpD domain of the bacterial bifunctional protein TrpGD.

[0065] The nucleic acid sequences encoding TrpG can be selected from the group consisting of nucleic acid sequences encoding the TrpG domain of the Escherichia coli TrpGD protein (positions 3 to 196 of SEQ ID NO: 1), TrpG of Bacillus subtilis (Uniprot accession number: P28819; SEQ ID NO: 2), the TrpG domain of the Salmonella typhimurium TrpGD protein (Uniprot accession number: P00905; position 3 to 196 of SEQ ID NO: 3), TrpG of Cupriavidus necator (Uniprot accession number: Q0K6I2; SEQ ID NO: 4), and TrpG of Corynebacterium glutamicum (Uniprot accession number: P06558; SEQ ID NO: 5). TrpG polypeptides can also be any polypeptide that exhibits TrpG activity and has at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the sequences of SEQ ID NOs. 2, 4, and 5, SEQ ID NOs. 1 from position 3 to position 196, and SEQ ID NOs.

[0066] Nucleic acid sequences encoding TrpD can be selected from the group consisting of nucleic acid sequences encoding the TrpD domain of the Escherichia coli (K12 strain) TrpGD protein (positions 202 to 531 of SEQ ID NO: 1), TrpD of Bacillus subtilis (Uniprot accession number: P03947; SEQ ID NO: 6), the TrpD domain of the Salmonella tyhumurium TrpGD protein (Uniprot accession number: P00905; position 202 to 531 of SEQ ID NO: 3), TrpD of Capriavidus necatol (Uniprot accession number: Q0K6I1; SEQ ID NO: 7), and TrpD of Corynebacterium glutamicum (Uniprot accession number: P06559; SEQ ID NO: 8). TrpD polypeptides can also be any polypeptide that exhibits TrpD activity and has at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the sequences of SEQ ID NOs. 6, 7, and 8, SEQ ID NOs. 1 from position 202 to 531, and SEQ ID NOs.

[0067] Other TrpD and TrpG polypeptides can be easily identified using routine methods, for example, based on their homology to the polypeptides listed above.

[0068] Preferably, the TrpD polypeptide and the TrpG polypeptide are derived from the same bacterial species. More preferably, the TrpD polypeptide and the TrpG polypeptide are derived from the same species as the recombinant bacteria of the present invention.

[0069] The recombinant bacteria of the present invention have been genetically modified to induce an imbalance between anthranylate phosphoribosyltransferase activity and anthranylate synthase activity, prioritizing anthranylate synthase activity.

[0070] In some embodiments, this imbalance is induced by adjusting the expression levels of TrpD and TrpG and / or TrpE, preferably the expression level of TrpG, more preferably the expression levels of TrpG and TrpE. In fact, in these embodiments, the expression level of TrpD is lower than the expression level of TrpG and / or TrpE, preferably lower than the expression level of TrpG, and more preferably lower than the expression levels of TrpG and TrpE. In particular, the TrpD expression level is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the TrpG or TrpE expression level, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the TrpG expression level, more preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the TrpG expression level, and at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the TrpE expression level.

[0071] The expression level of a polypeptide can be determined by various techniques. In particular, the expression level can be determined by measuring the amount of the polypeptide or the corresponding mRNA. Preferably, the expression level is determined by measuring the amount of the corresponding mRNA. Methods for determining the amount of mRNA are well known in the art. For example, nucleic acids contained in bacteria are first extracted according to standard methods, for example, using lytic enzymes or chemical solutions, or by nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR). Preferably, the mRNA corresponding to the polypeptide is detected and quantified by quantitative RT-PCR or semi-quantitative RT-PCR. Real-time quantitative RT-PCR or semi-quantitative RT-PCR is particularly advantageous. Primer pairs are designed to specifically detect and quantify the polypeptide of interest.

[0072] The recombinant bacteria of the present invention may include a gene encoding TrpG under the control of a first promoter and a gene encoding TrpD under the control of a second promoter. The first and second promoters may be two different promoters exhibiting different transcription intensities / rates. Preferably, the first promoter is a promoter that is not operably ligated to the gene encoding TrpG in the unmodified bacteria, and / or the second promoter is a promoter that is not operably ligated to the gene encoding TrpD in the unmodified bacteria.

[0073] In certain embodiments, the recombinant bacteria of the present invention are - The gene encoding TrpG or the first operon under the control of the first promoter, Here, the first operon includes at least a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding TrpE. - The gene encoding TrpD under the control of the second promoter or the second operon, Here, the second operon includes at least a nucleic acid sequence encoding TrpD. Includes.

[0074] In the above embodiment, the recombinant bacterium of the present invention comprises recombinant nucleic acid including a gene encoding TrpG or a first operon under the control of a first promoter, and / or recombinant nucleic acid including a gene encoding TrpD or a second operon under the control of a second promoter. Preferably, the first promoter is a promoter that is not operably linked to the gene encoding TrpG in the unmodified bacterium, and / or the second promoter is a promoter that is not operably linked to the gene encoding TrpD in the unmodified bacterium.

[0075] The first or second operon, preferably the second operon, may further include a nucleic acid sequence encoding indole-3-glycerol phosphate synthase (TrpC) and / or a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase (TrpF) and / or a nucleic acid sequence encoding the α subunit (TrpA) of tryptophan synthase and / or a nucleic acid sequence encoding the β subunit (TrpB) of tryptophan synthase.

[0076] The genes encoding TrpG and TrpD, or the first and second operons, may be contained in one or more recombinant nucleic acids comprising one or more expression cassettes. Preferably, the expression cassette used in the present invention comprises at least a promoter operably ligated to the gene or operon and a transcription terminator that is recognized by bacteria and whose transcription is terminated. The terminator is operably ligated to the 3' end of the encoding nucleic acid. Any terminator that functions in a host cell can be used in the present invention and can be readily selected by those skilled in the art. Typically, the terminator is selected in relation to the promoter.

[0077] In one embodiment, the recombinant bacterium of the present invention comprises a first recombinant nucleic acid comprising a first expression cassette comprising a gene encoding TrpG or a first operon as defined above, and a second recombinant nucleic acid comprising a second expression cassette comprising a gene encoding TrpD or a second operon as defined above.

[0078] In another embodiment, the recombinant bacterium of the present invention comprises a recombinant nucleic acid comprising a first expression cassette containing a gene encoding TrpG or a first operon as defined above, and a second expression cassette containing a gene encoding TrpD or a second operon as defined above.

[0079] A first operon comprising a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding TrpE may be a chromosomal endogenous operon modified to inactivate or not express TrpD. In this case, the promoter controlling the expression of the operon may be a promoter controlling the expression of TrpG and optionally TrpE in unmodified bacteria.

[0080] In some embodiments, particularly when the unmodified bacteria express two distinct polypeptides, one exhibiting TrpD activity and the other exhibiting TrpG activity, the expression levels of TrpG and / or TrpE, preferably TrpG and TrpE, in the recombinant bacteria of the present invention are higher than the expression level of TrpD. Optionally, the expression levels of TrpG and / or TrpE, preferably TrpG and TrpE, may also be higher than the expression levels of TrpC, TrpF, TrpA and / or TrpB, preferably TrpD, TrpF, TrpA, TrpC and TrpB.

[0081] In some other embodiments, particularly when the non-modified bacteria express a bifunctional protein exhibiting TrpD activity and TrpG activity, the expression levels of TrpG and / or TrpE, preferably TrpG and TrpE, in the recombinant bacteria of the present invention may be higher, lower, or substantially equal to the expression level of TrpD, preferably higher or substantially equal. In fact, we have shown that recombinant bacteria genetically modified to separately express a polypeptide exhibiting glutamine amide transferase activity (TrpG) (and not anthranilate phosphoribosyltransferase activity) and a polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD) (and not glutamine amide transferase activity) accumulate anthranilic acid even when TrpD expression is controlled by a strong promoter.

[0082] The expression level of a gene or operon can be altered by any method known to those skilled in the art, particularly by adjusting the copy number of the gene or operon and / or adjusting the strength of the promoter that controls its expression.

[0083] The promoters used in the present invention may be any polynucleotides that exhibit transcriptional activity in host cells, including mutant promoters, cleavage promoters, and hybrid promoters, and can be obtained from endogenous or heterologous genes. Promoters used to control the expression of the gene encoding TrpD or the operon containing said gene, and the gene encoding TrpG or the operon containing said gene, may be constitutive or inductive. In particular, the activity of the promoter that controls the expression of the gene encoding TrpD or the operon containing said gene can be regulated by the tryptophan concentration.

[0084] In a preferred embodiment, the promoter controlling the expression of the gene encoding TrpG or the operon containing said gene is more potent than the promoter controlling the expression of the gene encoding TrpD or the operon containing said gene. In particular, the promoter controlling the expression of the gene encoding TrpG or the operon containing said gene may be a potent promoter, i.e., a promoter that results in a high transcription initiation rate, while the promoter controlling the expression of the gene encoding TrpD or the operon containing said gene may be a weak promoter, i.e., a promoter that results in a low transcription initiation rate. Alternatively, both promoters may be potent or weak promoters, provided that the imbalance between anthranilate phosphoribosyltransferase activity and anthranilate synthase activity maintains the dominance of anthranilate synthase activity. Examples of such potent promoters include, but are not limited to, the T7 promoter, pTac, pRecA, and pTrp. Examples of such weak promoters include, but are not limited to, pLac and pLacUV5. Further promoters are described in Sambrook et al., 2012; Molecular cloning: a laboratory manual, 4th edition, Cold Spring Harbor. In certain embodiments, the promoter controlling the expression of the gene encoding TrpG or the operon containing said gene is pTrp or a promoter of similar strength, preferably pTrp, and the promoter controlling the expression of the gene encoding TrpD or the operon containing said gene is pTac or a promoter of similar strength, preferably pTac.

[0085] In more specific embodiments, recombinant bacteria, preferably wild-type bacteria corresponding to Escherichia coli, express bifunctional proteins exhibiting TrpG activity and TrpD activity, wherein the recombinant bacteria are genetically modified to separately express a polypeptide exhibiting glutamine amide transferase activity (TrpG) (and not anthranilate phosphoribosyltransferase activity) under the control of a first promoter, and a polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD) (and not glutamine amide transferase activity) under the control of a second promoter, both of which are strong promoters. Preferably, the promoter controlling the expression of the gene encoding TrpG or the operon containing said gene is pTrp or a promoter of similar strength, preferably pTrp, and the promoter controlling the expression of the gene encoding TrpD or the operon containing said gene is pTac or a promoter of similar strength, preferably pTac.

[0086] Optionally, the recombinant nucleic acids used in the present invention may also include selection markers that enable easy selection of recombinant host cells. Typically, the selection marker is a gene encoding antibiotic resistance.

[0087] Bacteria can be transformed using expression vectors containing recombinant nucleic acids as disclosed above. The selection of an expression vector is typically determined by the compatibility of the vector with the host cell into which it is introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates together with the integrated chromosome. Preferably, a vector or a portion thereof containing at least one recombinant nucleic acid disclosed herein is inserted into the bacterial genome. If integration into the host cell genome occurs, the integration of the sequence into the genome may depend on homologous or non-homologous recombination. On the one hand, the vector may contain additional polynucleotides to direct integration by homologous recombination at a precise location in the host cell genome. These additional polynucleotides may be any sequence homologous to the target sequence in the host cell genome. On the other hand, the vector may be integrated into the host cell genome by non-homologous recombination. For autonomous replication, the vector may further include an origin of replication that enables the vector to autonomously replicate within the host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication and functions within the cell. The vector also preferably includes one or more selection markers that enable easy selection of the host cell containing the vector. Typically, the selection markers are genes encoding antibiotic resistance. Methods for selecting these elements by the host cell are well known to those skilled in the art. The expression vector can be constructed by classical techniques of molecular biology well known to those skilled in the art. The recombinant nucleic acid contained in the recombinant bacterium of the present invention may be integrated into the bacterial genome or maintained in episomal form within one or more expression vectors. In embodiments in which the recombinant nucleic acid is maintained in episomal form, the expression vector may be present in the bacterium in one or more copies, depending on the nature of the origin of replication. Preferably, the recombinant nucleic acid is integrated into the bacterial genome.One or more copies of the recombinant nucleic acid can be introduced into the genome by recombinant methods known to experts in the field, including gene substitution. Preferably, in embodiments in which the second recombinant nucleic acid is maintained in an expression vector in episomal form, the expression vector is present in the bacteria in fewer than 25 copies, more preferably fewer than 10 copies.

[0088] In certain embodiments, the recombinant bacterium of the present invention comprises a first recombinant nucleic acid, which is incorporated into the genome and comprises a first expression cassette containing a gene encoding TrpG or a first operon as defined above, and a second recombinant nucleic acid, which comprises a second expression cassette containing a gene encoding TrpD or a second operon as defined above, and is maintained in the form of an episome within an expression vector.

[0089] Recombinant bacteria can be further genetically modified to increase the carbon flux to anthranilic acid.

[0090] In particular, the recombinant bacteria of the present invention can be genetically modified to express heterologous genes encoding feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase enzyme. The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase, EC 4.1.2.15) catalyzes the condensation of phospho(enol)pyruvic acid (PEP) and erythritol-4-phosphate (E4P) to DAHP and inorganic phosphate. This reaction is the first confirmed step in the biosynthesis of aromatic compounds in microorganisms. Escherichia coli has three types of DAHP synthase isoenzymes, each of which is feedback-controlled by one of the aromatic amino acids tyrosine, phenylalanine, and tryptophan. As used herein, the terms “feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase” or “feedback-resistant DAHP synthase” refer to DAHP synthases that are not negatively controlled by the products of the schimate pathway, i.e., tyrosine-, phenylalanine-, and / or tryptophan-insensitive DAHP synthases. Feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases may be any feedback-resistant DAHP synthase known to those skilled in the art, preferably a bacterial feedback-resistant DAHP synthase.Examples of feedback-resistant DAHP synthases, though not limited to them, include: E. coli AroF with the substitution of Asn-8 to Lys-8 (Jossek et al. FEMS Microbiol Lett, August 7, 2001; 202(1): pp. 145-148), AroG with the substitution of Ala-146 to Ans-146 (Mascarenhas et al. Applied and Environmental Microbiology, October 1991, 57; 10: pp. 2995-2999), and AroH with the substitution of Pro-18 to Lys-18, Val-147 to Met-147, Gly-149 to Asp-149, Gly-149 to Cys-149, or Ala-177 to Tyr-177 (Ray et al. Journal of Bacteriology, December 1988). 170(12) pp. 5500-5506 is one example.

[0091] Alternatively, or further, the recombinant bacteria of the present invention may be genetically modified to overexpress an endogenous gene encoding a transketolase enzyme, or to express a heterologous gene encoding a transketolase enzyme. Transketolase (EC 2.2.1.1) catalyzes the reversible transfer of ketol groups between several donor and acceptor substrates. This enzyme reversibly links glycolysis and the pentose phosphate pathway. This enzyme is involved in the catabolism of pentose sugars and the supply of erythrose-4-phosphate (E4P), a precursor of aromatic amino acids. Escherichia coli contains two transketolase isozymes, TktA and TktB. Preferably, the recombinant bacteria of the present invention are genetically modified to overexpress an endogenous gene encoding a transketolase enzyme.

[0092] The recombinant nucleic acids or expression vectors disclosed above can be introduced into bacteria by any method known to those skilled in the art, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, or protoplast fusion. Depending on the properties of the host cell, those skilled in the art can easily select an appropriate method.

[0093] The recombinant bacteria of the present invention are preferably obtained by genetic modification of bacteria having the metabolic ability to synthesize tryptophan. In particular, the recombinant bacteria of the present invention are preferably obtained from bacteria having the metabolic ability to synthesize tryptophan, and the endogenous TrpG activity and TrpD activity in unmodified bacteria are expressed as the bifunctional protein TrpGD.

[0094] Recombinant bacteria may be any Gram-positive or Gram-negative bacteria. Suitable examples of bacteria include, but are not limited to, the genera Escherichia (e.g., Escherichia coli), Streptomyces, Bacillus, Cupridavidus, Corynebacterium, Mycobacterium, Kitasatospora, and Luteipulveratus. The genera include *Us*, *Thermobifida*, *Thermomonospora*, *Frankia*, *Pseudonocardia*, *Saccharothrix*, *Kutzneria*, *Lentzea*, *Prauserella*, *Salinispora*, and *Micromonospora*. The genera include Onospora, Actinoplanes, Catenulispora, Mycolicibacterium, Dietzia, Aeromicrobium, Nonomuraea, Blastococcus, Modestobacter, and Saccharopolyspora. Examples of bacteria include those belonging to the genera Aropolyspora, Amycolatopsis, Actinopolyspora, Acidimicrobium, Photorhabdus, Hoeflea, Azospirillum, Crinalium, or Cylindrospermum. Preferably, the recombinant bacteria of the present invention are selected from bacteria of the genera Escherichia, Streptomyces, Corynebacterium, and Bacillus.More preferably, the recombinant bacteria of the present invention are selected from bacteria of the genera Escherichia, Streptomyces, and Bacillus. Even more preferably, the recombinant bacteria of the present invention are Escherichia coli.

[0095] In certain embodiments, the recombinant bacterium is a genetically modified E. coli bacterium comprising a nucleic acid sequence encoding glutamine amide transferase (TrpG) under the control of a first promoter and a nucleic acid sequence encoding anthranilate phosphoribosyltransferase (TrpD) under the control of a second promoter. Preferably, the recombinant bacterium comprises (i) a gene or first operon encoding glutamine amide transferase (TrpG) under the control of a first promoter, wherein the first operon comprises at least a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding anthranilate synthase (TrpE); and (ii) a gene or second operon encoding anthranilate phosphoribosyltransferase (TrpD) under the control of a second promoter. The operon comprises a gene encoding TrpD or a second operon, wherein the second operon comprises at least a nucleic acid sequence encoding TrpD, and optionally a nucleic acid sequence encoding indole-3-glycerol phosphate synthase enzyme (TrpC), a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase enzyme (TrpF), a nucleic acid sequence encoding the α subunit of tryptophan synthase enzyme (TrpA), and / or a nucleic acid sequence encoding the β subunit of tryptophan sinter enzyme (TrpB). Both promoters can be strong promoters. Preferably, the promoter controlling the expression of the gene encoding TrpG or the operon containing the gene is pTrp or a promoter of similar strength, preferably pTrp, and the promoter controlling the expression of the gene encoding TrpD or the operon containing the gene is pTac or a promoter of similar strength, preferably pTac.

[0096] Preferably, the recombinant bacteria of the present invention do not exhibit salicylate 5-hydroxylase (S5H) activity and / or do not produce 5-hydroxyanthranilate (5-HAA).

[0097] Preferably, the recombinant bacteria of the present invention can produce at least 1 g / L of anthranilate when cultured in a 2 L fed-batch fermenter for 48 hours in the presence of glucose as a carbon source, or at least 4 g / L of anthranilate when cultured in a 20 L fed-batch fermenter. Alternatively, the recombinant bacteria of the present invention can produce at least 300 mg / L, preferably at least 400 mg / L, and more preferably at least 500 mg / L, when cultured in a 5 mL culture for 30 hours in the presence of glucose as a carbon source.

[0098] In a further embodiment, the present invention relates to the use of recombinant bacteria of the present invention for the production of anthranilic acid. The present invention also relates to a method for producing anthranilic acid, comprising the steps of culturing recombinant bacteria of the present invention under conditions suitable for producing the compound, and optionally recovering the anthranilic acid. Anthranilic acid is secreted by bacteria. Therefore, anthranilic acid can be recovered by taking a culture medium, in particular the culture supernatant. The method may further include the steps of isolating or purifying the anthranilic acid. Anthranilic acid can be isolated or purified using any method known to those skilled in the art, such as precipitation, ion exchange, reverse osmosis and filtration.

[0099] Preferably, the recombinant bacteria used in the method of the present invention do not produce 5-hydroxyanthranilate (5-HAA), and the anthranilic acid produced and optionally recovered lacks 5-HAA.

[0100] All of the above embodiments relating to recombinant bacteria of the present invention are also intended to be contemplated in these embodiments.

[0101] The conditions suitable for producing anthranilic acid can be easily determined by those skilled in the art depending on the recombinant bacteria used. In particular, those skilled in the art can easily select appropriate culture media and growth conditions depending on the bacteria. Preferably, the culture medium used to produce anthranilic acid using the recombinant bacteria of the present invention is deficient in tryptophan or any tryptophan source, or is not supplemented with tryptophan.

[0102] Further aspects and advantages of the present invention will be described in the following examples, which should be considered illustrative and not limiting. [Examples]

[0103] (Example 1) Materials and methods Strain construction The K1 strain was constructed by replacing the anthranilate phosphoribosyltransferase domain of trpD in E. coli strain MG1655 with a chloramphenicol cassette using the lambda red method (Kirill et al. PNAS, June 2000, 97 (12) pp. 6640-6645). Briefly, the chloramphenicol cassette of the pKD3 plasmid (Kirill et al. PNAS, June 2000, 97 (12) pp. 6640-6645) was amplified using primers 1 and 2 containing sequences homologous to the trpD gene (see Table 2 (Table 1) below). Subsequently, MG1655 cells were transformed with the pKD46 plasmid (Kirill et al. PNAS, June 2000, 97 (12) pp. 6640-6645) and its PCR product, thereby enabling lambda red recombination.

[0104] The K2 strain was constructed by removing the chloramphenicol cassette from the K1 strain using the pCP20 plasmid (Kirill et al., PNAS, June 2000, 97 (12) pp. 6640-6645). The K2 strain was then transformed with the p002 plasmid (see below) to obtain the K3 strain.

[0105] Plasmid construction The low-copy plasmid pBAC-LacZ (addgene n°13422) was used as the backbone. The lacZ gene was removed by digesting the pBAC-LacZ plasmid with SalI to obtain the pBAC-Δ(LacZ) plasmid. Next, the pTAC promoter (a hybrid of the E. coli trp promoter and the lac promoter) and the rrnB T1 terminator (a transcription terminator T1 derived from the E. coli rrnB gene) were added to this plasmid by digestion / ligation (SalI / HpaI and SalI / ZraI). The anthranilate phosphoribosyltransferase domain of trpD was amplified from the E. coli chromosome using primers 3 and 4 (see Table 2 (Table 1) below), and then cloned into pBAC-Δ(LacZ) between the pTAC promoter and the rrnB T1 terminator by digestion / ligation (NheI / HindIII) to obtain plasmid p002.

[0106] [Table 1]

[0107] Culture conditions K2 and K3 strains were cultured in 5 ml of LB medium for 8 hours, then cultured overnight in 10 ml of inorganic medium M9 (the composition of the medium is shown in Table 3 (Table 2)). 20 mg / l of tryptophan was added to the K2 culture. 100 μl of each pre-culture was used and inoculated into 25 ml of inorganic medium M9 under the same conditions as the pre-culture. The strains were then cultured at 37°C for 31 hours.

[0108] [Table 2]

[0109] analysis The amount of anthranilates in the culture supernatant was measured using the LC-UV method. The HPLC system was equipped with a Waters Acquity BEH C18 column (50 × 2.1 mm; 1.7 μm) and connected to a UV detector [190~400 nm]. An H2O / ACN solution was used as the mobile phase at 40°C, with a flow rate of 0.5 ml / , a linear elution gradient from 5% to 100% ACN over 8 minutes, and a UV detector (λ210 nm).

[0110] result K2 strain cultured in M9 medium without tryptophan did not grow and did not produce anthranilates. K2 strain cultured in M9 medium supplemented with tryptophan showed an optical density (OD) of 600 nm. 600nm The molecule grew until its concentration reached 6.3, producing 311 mg / l of anthranilic acid in 31 hours.

[0111] K3 strain cultured in tryptophan-free M9 medium had an OD of 6.8 600nm It proliferated to produce 375 mg / l of anthranilate in 31 hours. Therefore, strain K3 is a bacterial strain that can accumulate and secrete anthranilates without a tryptophan nutrient requirement.

[0112] (Example 2) Materials and methods Culture conditions - 2L-fed-batch fermentation Fed-batch fermentation was performed in a 2L bioreactor Sartorius® Steim Biostat C containing 1.35L of M9 culture medium supplemented with 30g / L glucose, 1M IPTG, and 50μg / L chloramphenicol. Cells were inoculated from cryostock (strains stored at -80°C in the presence of 20% glycerol) into 100mL of LB medium containing 1M IPTG and 50μg / L chloramphenicol in a 1L baffled flask at 37°C in a 200rpm rotary shaker for 8 hours. Subsequently, 2mL aliquots of seed culture were inoculated into a 2L baffled flask containing 200mL of M9 medium supplemented with 30g / L glucose, 1M IPTG, and 50μg / L chloramphenicol. Cells were cultured at 37°C and 200 rpm until the OD600 reached approximately 4, then transferred to a fermenter to achieve an initial OD600 of 0.2. The culture pH was controlled to 6.8 by automatically supplying 17% (v / v) ammonia solution or 2M H3PO4, and the temperature was maintained at 37°C. The dissolved oxygen concentration (DO) was controlled to 20% of the air saturation.

[0113] Biomass was monitored by measuring OD at 600 nm. Extracellular concentrations of anthranilates were determined by HPLC-UV 48 hours after culturing.

[0114] Culture conditions - 20L - fed-batch fermentation Fed-batch fermentation was performed in a 20L bioreactor, Sartorius® Stedim Biostat C, containing 16L of M9 culture medium supplemented with 30g / L glucose, 1M IPTG, and 50μg / L chloramphenicol. Cells were inoculated from 100μL of cryostock into 100mL of LB medium containing 1M IPTG and 50μg / L chloramphenicol in a 1L baffled flask and cultured at 37°C in a rotary shaker at 200rpm for 8 hours. Subsequently, 2×50mL of seed culture was inoculated into a 2×5L baffled flask containing 2×1L of M9 medium supplemented with 30g / L glucose, 1M IPTG, and 50μg / L chloramphenicol. Cells were cultured at 37°C at 200rpm until the OD600 reached approximately 4, then transferred to a fermenter to achieve an initial OD600 of 0.2. The culture pH was controlled to 6.8 by automatically supplying 35% (v / v) ammonia solution or 2M H3PO4, and the temperature was maintained at 37°C. The dissolved oxygen concentration (DO) was controlled to 20% of air saturation by automatically increasing the supplied air from 0.2 to 1 vvm and changing the stirring speed up to 1800 rpm. During culture, the residual glucose concentration was maintained between 10 and 30 g / L.

[0115] Biomass was monitored by measuring OD at 600 nm. Extracellular concentrations of anthranilates were determined by HPLC-UV 48 hours after culturing.

[0116] result The anthranilate production of strain K3 in 2L and 20L fed-batch fermenters is shown in Table 4 (Table 3) below.

[0117] [Table 3]

[0118] (Example 3) Materials and methods Strain construction The K2 strain disclosed in Example 1 was modified to overexpress the endogenous tktA enzyme by replacing the native promoter of the tktA gene encoding the transketolase enzyme with a stronger promoter, thereby obtaining the K51 strain.

[0119] Next, strain K51 was further manipulated to (i) replace the native promoter of the aroG gene encoding the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase enzyme with a stronger promoter, and (ii) directly mute the endogenous aroG gene on the chromosome by introducing a mutation that results in a feedback-resistant aroG enzyme. In this regard, the aroG gene from E. coli was amplified and cloned into a plasmid by restriction / ligation. Then, mutagenesis was performed on this plasmid using the QuickChange method to change aroG to aroG fbr The (G436A) mutation was introduced. Finally, this substituted promoter was subjected to PCR to aroG fbr It was cloned into a plasmid that could be inserted before the gene and enable recombination with the chromosome. Then, under the control of a promoter stronger than the native promoter, this aroG fbr A recombinant cassette containing the gene was introduced into strain K51 to obtain strain K77. Then, strain K77 was transformed with the p002 plasmid to obtain strain K91.

[0120] Culture conditions The K91 strain was cultured in 5 ml of LB medium for 8 hours, and then cultured overnight in 10 ml of inorganic medium M9 (the medium composition is shown in Table 3 (Table 4)). 20 mg / l of tryptophan was added to the K2 culture. 100 μl of each pre-culture was inoculated into 25 ml of inorganic medium M9 under the same conditions as the pre-culture. The strains were then cultured at 37°C for 3 hours.

[0121] analysis The amount of anthranilates in the culture supernatant was measured using the LC-UV method. The HPLC system was equipped with a Waters Acquity BEH C18 column (50 × 2.1 mm; 1.7 μm) and connected to a UV detector [190~400 nm]. An H2O / ACN solution was used as the mobile phase at 40°C, with a flow rate of 0.5 ml / , a linear elution gradient from 5% to 100% ACN over 8 minutes, and a UV detector (λ210 nm).

[0122] result K91 strain cultured in tryptophan-free M9 medium grew to an OD of 7.0 at 600 nm and produced 573 mg / l of anthranilate in 32 hours. Therefore, K91 strain is a bacterial strain capable of accumulating and secreting anthranilates without a tryptophan nutritional requirement.

Claims

1. A method for producing anthranilic acid, comprising the steps of culturing recombinant bacteria and optionally recovering anthranilic acid, wherein the non-modified bacteria corresponding to the recombinant bacteria are Escherichia coli bacteria expressing a bifunctional protein exhibiting TrpG activity and TrpD activity, the recombinant bacteria are genetically modified to individually express (i) a polypeptide exhibiting glutamine amide transferase activity (TrpG) and not anthranilate phosphoribosyltransferase activity, and (ii) a polypeptide exhibiting anthranilate phosphoribosyltransferase activity (TrpD) and not glutamine amide transferase activity, and the recombinant bacteria are controlled by (i) a gene encoding glutamine amide transferase (TrpG) or a first promoter. A recombinant nucleic acid comprising (ii) a gene encoding anthranilate phosphoribosyltransferase (TrpD) or a second operon under the control of a second promoter, wherein the second operon comprises a nucleic acid sequence encoding at least TrpG and optionally an anthranilate synthase (TrpE) sequence, and a method comprising (ii) a recombinant nucleic acid comprising a gene encoding anthranilate phosphoribosyltransferase (TrpD) or a second operon, wherein the second operon comprises a nucleic acid sequence encoding at least TrpD, wherein the first promoter is stronger than the second promoter, thereby reducing the ratio of anthranilate phosphoribosyltransferase activity to anthranilate synthase activity compared to the unmodified bacteria, and enabling the recombinant bacteria to grow in tryptophan-deficient culture medium.

2. The method according to claim 1, wherein the recombinant bacterium is genetically modified to suppress the expression of the TrpD domain of the endogenous bifunctional protein TrpGD.

3. The method according to claim 1 or 2, wherein the recombinant bacteria are genetically modified to suppress the expression of the endogenous bifunctional protein TrpGD.

4. The method according to any one of claims 1 to 3, wherein the first promoter is a promoter that is not operably linked to the gene encoding TrpG in the unmodified bacterium, and / or the second promoter is a promoter that is not operably linked to the gene encoding TrpD in the unmodified bacterium.

5. The method according to any one of claims 1 to 4, wherein the second operon further comprises a nucleic acid sequence encoding indole-3-glycerol phosphate synthase enzyme (TrpC), a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase enzyme (TrpF), a nucleic acid sequence encoding the α subunit of tryptophan synthase enzyme (TrpA), and / or a nucleic acid sequence encoding the β subunit of tryptophan synthase enzyme (TrpB).

6. The method according to any one of claims 1 to 5, wherein the recombinant bacteria are also genetically modified to express a gene encoding a feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase enzyme, and / or to overexpress an endogenous gene encoding a transketolase enzyme, or to express a heterologous gene encoding a transketolase enzyme.

7. The method according to any one of claims 1 to 6, wherein the recombinant bacteria are cultured in a culture medium lacking tryptophan or any tryptophan source.

8. The method according to any one of claims 1 to 7, wherein the recombinant bacteria can produce at least 1 g / L of anthranilate when cultured in a 2 L fed-batch fermenter for 48 hours in the presence of glucose as a carbon source, or can produce at least 4 g / L of anthranilate when cultured in a 20 L fed-batch fermenter.

9. Recombinant bacteria as defined in any one of claims 1 to 8.

10. (i) A gene or first operon encoding glutamine amide transferase (TrpG), under the control of the first promoter, wherein the first operon comprises at least a nucleic acid sequence encoding TrpG and optionally a nucleic acid sequence encoding anthranilate synthase (TrpE); and (ii) A gene or second operon encoding anthranilate phosphoribosyltransferase (TrpD), under the control of the second promoter, wherein Recombinant bacteria according to claim 9, comprising a gene encoding TrpD or a second operon, the second operon comprising at least a nucleic acid sequence encoding TrpD and optionally a nucleic acid sequence encoding indole-3-glycerol phosphate synthase enzyme (TrpC), a nucleic acid sequence encoding phosphoribosyl anthranylate isomerase enzyme (TrpF), a nucleic acid sequence encoding the α subunit of tryptophan synthase enzyme (TrpA), and / or a nucleic acid sequence encoding the β subunit of tryptophan synthase enzyme (TrpB).

11. Use of the recombinant bacteria according to claim 9 or 10 for producing anthranilic acid.

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