Microorganism into which exogenous ribose-phosphate diphosphate kinase has been introduced, and method for producing L-tryptophan using same

Abstract

The present disclosure relates to: a Corynebacterium genus microorganism having an L-tryptophan producing ability, into which a ribose-phosphate pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the same has been introduced; a method for producing L-tryptophan comprising culturing the microorganism in a culture medium; a composition for producing L-tryptophan comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of the microorganism for the production of L-tryptophan.

Description

Technical Field

[0001] This disclosure relates to a Corynebacterium sp. microorganism capable of producing L-tryptophan, wherein the microorganism has been introduced with a ribo-phosphoro-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the phospho-pyrophosphate kinase; a method for producing L-tryptophan, comprising culturing the microorganism in a culture medium; a composition for producing L-tryptophan comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of the microorganism for producing L-tryptophan. Background Technology

[0002] The process of producing desired substances (such as amino acids) from microorganisms is an environmentally friendly and safe production method and has become the subject of various research. Among these, research on the large-scale production of desired substances from Corynebacterium microorganisms has been ongoing. Corynebacterium microorganisms, especially Corynebacterium glutamicum, are Gram-positive microorganisms widely used in the production of L-amino acids and other useful substances.

[0003] L-amino acids are the basic structural units of proteins and are important materials used in pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and fungicides. Various studies have been conducted to develop efficient production microorganisms and fermentation processes for the production of L-amino acids and other useful substances. For example, substance-specific methods are primarily employed, such as increasing the expression of genes encoding enzymes involved in L-tryptophan biosynthesis or eliminating genes unnecessary for biosynthesis (US8945907 B2).

[0004] L-Tryptophan is an essential amino acid that has been widely used as a raw material for pharmaceuticals and health foods, such as feed additives and infusion solutions. Although it can be produced through chemical synthesis, enzymatic methods, and fermentation, direct fermentation using microorganisms is currently the primary production method. Previous studies have demonstrated through intracellular quantitative analysis that, among 20 amino acids, tryptophan biosynthesis requires the highest level of energy (Proc. Natl. Acad. Sci. USA, (2002) Vol. 99, pp. 3695–3700). Therefore, further research is needed to effectively improve L-Tryptophan production capacity. Summary of the Invention

[0005] Technical issues

[0006] The technical problem to be solved by this disclosure is to provide a microorganism in which exogenous ribose-phospho-pyrophosphate kinase is introduced, and a method for producing L-tryptophan using the microorganism.

[0007] Technical solution

[0008] One aspect of this disclosure provides a Corynebacterium microorganism with L-tryptophan production capacity, wherein a ribo-phosphoro-pyrophosphate kinase or a polynucleotide encoding the ribo-phosphoro-phosphorokinase derived from Bacillus subtilis has been introduced into the microorganism.

[0009] In one specific embodiment, the ribo-phospho-pyrokinase may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity with it.

[0010] In another specific implementation, the ribo-phosphoro-pyrophosphate kinase derived from Bacillus subtilis can be encoded by the prsA gene.

[0011] In another specific embodiment, the polynucleotide encoding ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis may contain the base sequence of SEQ ID NO: 2.

[0012] In another specific embodiment, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0013] In any of the aforementioned specific embodiments, the Corynebacterium species may have increased L-tryptophan production capacity compared to unmodified microorganisms.

[0014] Another aspect of this disclosure provides a method for producing L-tryptophan, comprising culturing a Corynebacterium genus microorganism capable of producing L-tryptophan in a culture medium, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the ribo-phospho-pyrophosphate kinase.

[0015] In one specific implementation, the method may further include recovering L-tryptophan from cultured microorganisms, microbial cultures, microbial fermentation products, or culture media.

[0016] Another aspect of this disclosure provides a composition for producing L-tryptophan, comprising: a Corynebacterium microorganism having L-tryptophan production capability, said microorganism having incorporated a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding thereus; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more of these.

[0017] Beneficial effects

[0018] The present invention discloses a Corynebacterium microorganism capable of producing L-tryptophan, wherein the microorganism has been introduced with ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or the polynucleotide encoding it, which can produce L-tryptophan in high yield and is therefore effective for industrial production of L-tryptophan. Detailed Implementation

[0019] This disclosure will be described in detail below. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below. In addition, numerous papers and patent documents are cited throughout this specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0020] definition

[0021] As used in the specification and appended claims of this disclosure, the singular articles (“a,” “an,” and “the”) may include plural referents unless the context clearly indicates otherwise. Furthermore, unless the context clearly indicates otherwise, singular terms may include their plural forms, and plural terms may include their singular forms. Additionally, as used in the specification and appended claims of this disclosure, the use of “or” may include the meaning of “and / or” unless otherwise stated.

[0022] As used herein, the term "approximately" may precede a specific numerical value. As used herein, the term "approximately" includes not only the exact numerical value specified after the term, but also a range that approximates or is close to that value. Considering the context in which the number appears, it can be determined whether the specific number mentioned is close to or approximates that number. In one instance, the term "approximately" may refer to the range of -10% to +10% of a value. In another instance, the term "approximately" may refer to the range of -5% to +5% of a given value. However, it is not limited to these examples.

[0023] As used herein, terms such as “first, second, third…”, “i), ii), iii)…”, or “(a), (b), (c), (d)…” can be used to distinguish each component. When these terms are used in relation to the steps of a method, purpose, or analysis, they do not imply that the steps will be performed sequentially or in a particular order, and the steps may be performed, for example, without time intervals between steps, simultaneously or sequentially, in reverse order, or in a random order at intervals of seconds, minutes, hours, days, or months.

[0024] As used herein, the term "composed of" means that the total percentage of a specific feature, step, component, or other element in the term is 100%. The features, steps, components, or other elements in the term "composed of" may be necessary or mandatory. For example, any other features, steps, components, or other elements, or non-essential features, steps, components, or other elements, may be excluded in addition to those in the term "composed of".

[0025] As used herein, the term "consistently of" can mean that the feature, step, component or other element claimed in this disclosure may be present without being substantially affected by the presence of at least one unspecified feature, step, component or other element.

[0026] As used herein, the term "comprising" means the presence of a feature, step, component, or other element that follows the term, and does not exclude the presence of at least one additional feature, step, or component. As used herein, the specific feature, step, component, or other element that follows the term "comprising" may be necessary or mandatory; however, in some specific embodiments, other arbitrary or non-essential features, steps, components, or other elements may also be included.

[0027] Proteins, polypeptides

[0028] As used herein, the terms "protein" or "peptide" refer to a polymer or oligomer of consecutive amino acid residues. In this disclosure, the terms "peptide," "protein," and "peptide" are used interchangeably.

[0029] As used herein, the terms "mature polypeptide" or "mature protein" refer to a polypeptide or protein in the form that lacks a signal sequence or precursor peptide sequence. A mature polypeptide or mature protein can be the functional form of a polypeptide or protein. A mature polypeptide or mature protein refers to the final form of a polypeptide after translation and / or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N-terminal manipulation, C-terminal truncation, glycosylation, phosphorylation, leader sequence removal, etc.

[0030] In this disclosure, unless otherwise stated, the amino acid sequence is described in the direction from the N-terminus to the C-terminus.

[0031] In this disclosure, with regard to the amino acid sequence, a polypeptide or protein obviously "contains" the amino acid sequence shown in a specific sequence number, is "composed of" the amino acid sequence shown in a specific sequence number, or "has" the amino acid sequence shown in a specific sequence number. It may also contain polypeptides or proteins with certain amino acid deletions, modifications, substitutions, or additions, provided they have the same or corresponding activities as the polypeptide or protein composed of the amino acid sequence of that sequence number. For example, a polypeptide or protein may also contain, in its internal region or upstream or downstream region (N-terminus or C-terminus), polypeptides or proteins with amino acid additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions that do not alter the function of the proteins of this disclosure.

[0032] Furthermore, the range of polypeptides or proteins with the amino acid sequence shown in a specific sequence number may also include, for example, polypeptides or proteins conjugated to an N-terminal signal (or leader) sequence involved in the co-translation or post-translational translocation of a protein (polypeptide), or polypeptides or proteins conjugated to another sequence or linker, so as to enable the identification, purification or synthesis of the polypeptide or protein.

[0033] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) can include arginine, lysine, histidine, glutamic acid, and aspartic acid, while amino acids with uncharged side chains (also called uncharged amino acids or neutral amino acids) can include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. In yet another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids (branched amino acids). In another example, the 20 amino acids can be divided into five groups based on size, starting with the smaller groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the classification of amino acids is not limited to this. Generally, conserved substitutions have little or no effect on the activity of polypeptides or proteins.

[0034] Genes, polynucleotides

[0035] As used herein, the term "gene" narrowly refers to a polynucleotide that encodes a functional molecule, and broadly refers to a polynucleotide that contains a polynucleotide that encodes a functional molecule and a polynucleotide that contains upstream or downstream regions of that polynucleotide. In one specific embodiment, the functional molecule may be RNA or a protein, and the gene may have sequences (introns) inserted between each coding region (exons).

[0036] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleic acid molecule” refer to a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds to form a long chain, and refer to a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain having at least a certain length. In this disclosure, “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably.

[0037] Identity, homology

[0038] As used herein, the term “identity” or “homology” refers to the degree of similarity between two given amino acid sequences or base sequences, expressed as a percentage. In this disclosure, “homology” and “identity” are generally used interchangeably.

[0039] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, and a default gap penalty established by the program used can be applied together.

[0040] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using, for example, a known computer algorithm such as the “FASTA” program (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA85]: 2444) with default parameters. Alternatively, sequence information can be compared using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) to determine the sequence information, such as the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), or GAP computer programs such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J MOLEC BIOL 215:403 (1990); Guide to The results were obtained in HugeComputers, Martin J. Bishop (ed.), Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073. For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0041] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be confirmed by Southern hybridization under appropriate hybridization conditions, and appropriate hybridization conditions can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. For example, homologous or identical polynucleotide sequences can typically hybridize at least about 50%, 60%, 70%, 80%, or 90% along the entire sequence or full length under stringent conditions.

[0042] As used herein, the term “strict conditions” refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see Sambrook et al., ibid., 9.50–9.51, 11.7–11.8). For example, stringent conditions may include conditions under which polynucleotides with high homology or identity, i.e., polynucleotides with at least 60%, 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% homology or identity, hybridize with each other, while polynucleotides with homology or identity lower than the above-mentioned homology or identity do not hybridize with each other; or may include ordinary washing conditions for Southern hybridization, i.e., washing once, specifically twice, or three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0043] Hybridization can occur between nucleotides with complementary nucleotide sequences, but depending on the strictness of the hybridization, the hybridized polynucleotide may contain some base mismatches. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may comprise isolated nucleic acid fragments complementary to the entire sequence, as well as a substantially similar base sequence thereto.

[0044] For example, polynucleotides homologous to or identical to the polynucleotides disclosed herein can be tested at 55°C using a T... m The value is detected by hybridization. In addition, T... m The value can be 60°C, 63°C or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art.

[0045] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, variables that are well known in the field (e.g., Sambrook et al., ibid.).

[0046] Nucleic acid constructs, vectors, and transformation

[0047] As used herein, the term "nucleic acid construct" refers to an artificially designed single- or double-stranded nucleic acid molecule contained in a vector, which can be used to integrate desired genetic material into a suitable host or host cell. In one instance, the nucleic acid construct may contain a transgene delivered via a transformation vector, which enables the inserted sequence to replicate and / or be expressed in a host cell. In one instance, the transgene may be replicated from an existing sequence or may be artificially synthesized.

[0048] As used herein, the term "vector" refers to a DNA construct used to deliver desired polynucleotides into a suitable host or host cell.

[0049] In one example, the vector may contain a polynucleotide sequence encoding a desired polypeptide, operatively linked to a suitable expression regulatory region (or expression regulatory sequence) to enable expression of the desired polypeptide in a suitable host. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. After transformation into a suitable host cell (microorganism), the vector may replicate or function independently of the host genome, or it may integrate into the genome itself for replication or function.

[0050] Furthermore, in one instance, the vector of this disclosure may contain a sequence for inserting the desired polynucleotide into a chromosome. Insertion of a polynucleotide into a chromosome using a vector can be performed by any method known in the art, such as homologous recombination, but is not limited thereto.

[0051] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, granules, viruses, and bacteriophages in their native or recombinant states. For example, as phage vectors or granule vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., may be used; as plasmid vectors, those based on pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., may be used. In one example, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, etc., may be used.

[0052] The vector may further include selection markers to confirm transformation into host cells, or further, insertion into the chromosome of the host cell. Selection markers are used to select cells transformed with the vector or to confirm the insertion of desired polynucleotides into the chromosome; markers that confer selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface peptides, may be used. In an environment treated with a selector, only cells expressing the selection marker survive or exhibit a different phenotype, thus allowing for the selection of transformed cells.

[0053] As used herein, the term "transformation" refers to the introduction of a desired polynucleotide or a vector containing that polynucleotide into a host cell (microorganism), thereby altering the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into the chromosome of the host cell (microorganism) or located extrachromosomally. Furthermore, the polynucleotide may comprise DNA or RNA. Depending on the purpose of the introduction, the polynucleotide may be introduced in a suitable form. For example, a polynucleotide for expressing a desired polypeptide may be introduced into the host cell (microorganism) in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. The expression cassette may typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operatively linked to the coding sequence of the desired polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell (microorganism) as is and may be operatively linked to, but is not limited to, the sequence required for expression in the host cell (microorganism).

[0054] As used herein, the term "operably linked" refers to an arrangement in which a regulatory sequence is positioned appropriately to regulate the expression of a coding sequence. Therefore, the term "operably linked" includes attaching or linking a regulatory region (such as a promoter, stop codon, signal sequence, or enhancer region) with a functional domain having known or desired activity to a target (gene or polypeptide) to regulate the expression, secretion, or function of the target according to known or desired activity. For example, it might mean the functional linking of a polynucleotide sequence encoding a polypeptide to a promoter sequence that initiates and mediates polynucleotide transcription.

[0055] As used herein, the term “expression” includes, but is not limited to, any step involved in peptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0056] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule containing a desired polynucleotide sequence and an operatively linked regulatory sequence for its expression. For example, it may contain a base sequence of a polynucleotide encoding a desired polypeptide, operatively linked to a suitable expression regulatory region (or expression regulatory sequence) so that the desired polypeptide can be expressed in a suitable host.

[0057] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence essential for regulating the expression of a desired polynucleotide sequence. Each regulatory sequence can be a natural (from the same source) or exogenous (from a different gene) regulatory sequence of the coding sequence, a variant of that sequence, or another artificial sequence. Examples of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome-binding domains, and sequences regulating transcription and translation termination. The smallest unit of a regulatory sequence can contain a promoter as well as sequences that terminate transcription and translation.

[0058] As used in this article, the term “genetic recombination” refers to a natural or artificial process in which the elements that make up a gene, such as DNA or RNA, are rearranged into a sequence different from the original sequence during the process of decomposition and recombination.

[0059] As used herein, the term "recombinant gene" refers to a gene having a novel genomic structure resulting from genetic recombination (such as chemical synthesis or genetic engineering techniques). In this disclosure, the terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably. In one instance, the recombinant gene may comprise an artificial combination of nucleic acid fragments, such as regulatory sequences not naturally found together.

[0060] As used in this article, the term "recombinant protein" refers to a protein produced by genetic recombination.

[0061] microorganism

[0062] As used herein, the term "microorganism (or strain)" includes wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have undergone natural or artificial genetic modifications. It can be a microorganism whose specific mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and it can be a microorganism containing genetic modifications for the production of desired polypeptides, proteins, or products. In this disclosure, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0063] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a genotype and / or phenotype different from that of naturally occurring microorganisms (e.g., when the genetic modification affects how nucleic acid sequences are encoded in the microorganism), and may include all or potential offspring of that microorganism. In this disclosure, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not found in their natural (non-recombinant) form, not express genes expressed in their natural form, or express natural genes in a manner different from their natural expression.

[0064] For example, the microorganisms disclosed herein may be microorganisms that have introduced ribose-phospho-pyrokinase or polynucleotides encoding it (such as recombinant microorganisms), but are not limited thereto.

[0065] As used herein, the term "microorganism with L-tryptophan production capacity" refers to a microorganism capable of producing L-tryptophan in a living organism, and may include any microorganism that inherently lacks L-tryptophan production capacity but has been conferred the ability to produce L-tryptophan, as well as any microorganism that inherently possesses L-tryptophan production capacity. L-tryptophan production capacity can be conferred or enhanced through strain modification.

[0066] As used herein, the term "unmodified microorganism (strain)" does not exclude the inclusion of microorganisms (strains) that may have naturally occurring mutations, and may refer to wild-type microorganisms (strains) or natural microorganisms (strains) as is, or microorganisms (strains) before their traits are altered by genetic mutation due to natural or artificial factors. In this disclosure, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)," "unmutated microorganism (strain)," "parental microorganism," "parental strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard microorganism (strain)." The unmodified microorganism of this disclosure may refer to a microorganism (strain) in which ribo-phospho-pyrokinase or the polynucleotide encoding it is not introduced, or a microorganism (strain) before its introduction, but is not limited thereto. Additionally, in this disclosure, an unmodified microorganism may be a microorganism that does not contain a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or a polynucleotide consisting of the sequence of SEQ ID NO: 2, but is not limited thereto.

[0067] Increased protein (peptide) activity

[0068] As used herein, “increased” protein (peptide) activity refers to an increase in the activity of a protein (peptide) in a host cell (microorganism) compared to its inherent activity. This increase can be used interchangeably with terms such as “activation,” “upregulation,” “overexpression,” or “enhancement.” The host cell (microorganism) can be a prokaryotic or eukaryotic microorganism.

[0069] Increased protein (peptide) activity can include situations where the host cell (microorganism) exhibits protein (peptide) activity that it does not inherently possess, as well as situations where the host cell exhibits improved protein (peptide) activity compared to its inherent activity or its activity before modification.

[0070] For example, the case in which it “exhibits protein (peptide) activity that it does not inherently possess” or the case in which it exhibits improved protein (peptide) activity may be due to “the introduction of a protein (peptide),” but is not limited thereto.

[0071] As used herein, the term "introduction" of a protein (peptide) refers to the expression of a gene not originally present in the microorganism, thereby exhibiting the activity of a specific protein, or to an enhancement, increase, or improvement of peptide activity compared to its inherent activity or its activity before modification. For example, it can be achieved by introducing a gene encoding a protein (peptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (peptide) can be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (peptide) can be introduced into a host cell (microorganism), thereby exhibiting or enhancing its activity.

[0072] "Inherent activity" refers to the activity of a specific protein (peptide) that was originally present in the host cell (microorganism) before transformation or in the unmodified host cell (microorganism) when the trait is altered through genetic modification due to natural or artificial factors. This term is interchangeable with "unmodified activity".

[0073] An increase in protein (peptide) activity compared to its endogenous activity refers to an enhanced activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) compared to the activity and / or concentration (expression level) of the protein (peptide) originally present in the unmodified host cell (microorganism) or the unmodified host cell (microorganism).

[0074] In one instance, an increase may refer to the presence of the activity of a corresponding protein (peptide) that is not present, or an increase in the activity or concentration of a protein relative to the activity or concentration in the unmodified host cell (microorganism) or the unmodified host cell (microorganism), typically an increase of at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, up to at least about 1000% or at least about 2000% or more, but not limited thereto.

[0075] Increased protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or by increasing the activity of endogenous proteins (peptides). Whether protein (peptide) activity has increased can be confirmed by the increase in the protein (peptide) activity level, its expression level, or the amount of product generated by the activity of the corresponding protein (peptide).

[0076] Increased protein (peptide) activity can be achieved by a variety of methods well known in the art, and is not limited thereto, as long as the desired protein (peptide) activity can be increased compared to the activity of the host cell (microbe) before modification. Specifically, it can be achieved by using genetic engineering and / or protein engineering techniques well known to those skilled in the art, which are routine molecular biology techniques, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0077] Specifically, the increased protein (peptide) activity of this disclosure may be due to:

[0078] 1) Increase the intracellular copy number of the polynucleotide encoding the protein (peptide);

[0079] 2) Modify the gene expression regulatory region on the chromosome that encodes the protein (peptide) (e.g., introduce modifications to the expression regulatory region, replace it with a sequence that shows stronger activity, or insert a sequence that shows stronger activity).

[0080] 3) Modify the start codon or 5'-UTR base sequence of the gene transcript (which encodes the protein (polypeptide));

[0081] 4) Modify the amino acid sequence of the protein (peptide) to increase its activity;

[0082] 5) Modify the polynucleotide sequence encoding the protein (peptide) to increase the activity of the protein (peptide) (e.g., modify the polynucleotide sequence of the gene encoding the protein (peptide) so that the gene encodes a modified protein (peptide) with increased activity).

[0083] 6) Introduce an exogenous protein (peptide) exhibiting the activity of the protein (peptide), or an exogenous polynucleotide encoding the protein (peptide);

[0084] 7) Codon optimization of the polynucleotides encoding the protein (peptide);

[0085] 8) By analyzing the tertiary structure of the protein (peptide), select and modify or chemically modify the exposed regions of the protein (peptide);

[0086] 9) Regulate the cellular localization of the protein (peptide); or

[0087] 10) Selected from two or more of the above 1) to 9), but not particularly limited thereto.

[0088] For example,

[0089] 1) Increasing the intracellular copy number of the polynucleotide encoding the protein (peptide) can be achieved by introducing a vector containing the polynucleotide encoding the protein (peptide) into a host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Alternatively, this can be achieved by introducing one or two or more copies of the polynucleotide encoding the protein (peptide) into the chromosome of the host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Chromosomal introduction can be performed by introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome, but is not limited thereto. The vector is as described above. Regarding the polynucleotide sequence encoding the protein, the regulatory sequence can be a natural sequence (of the same origin) or a foreign sequence (from a different gene), a variant thereof, or another artificial sequence, and can induce the expression of the polynucleotide in the host cell (microorganism).

[0090] 2) Replacing the expression regulatory region (or expression regulatory sequence) of the gene encoding the protein (polypeptide) on the chromosome with a highly active sequence can be achieved, for example, by introducing modifications into the sequence via deletion, insertion, substitution, or a combination thereof to further increase the activity of the expression regulatory region, or by replacing the sequence with a more active sequence. The expression regulatory region may include, but is not particularly limited to, promoters, operon sequences, sequences encoding ribosome binding sites, sequences regulating transcription and translation termination, etc. In one instance, it may specifically include replacing the original promoter with a strong promoter, but is not limited thereto.

[0091] Examples of known strong promoters include the cj1 to cj7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, yccA promoter, etc., but strong promoters are not limited to these.

[0092] 3) Modifying the start codon or 5'-UTR of a gene (which encodes the protein (peptide)) can be achieved, for example, by modifying the base sequence to encode a different start codon that has a higher protein (peptide) expression rate compared to the endogenous start codon, or by modifying the base sequence to encode a ribosome binding site (RBS) sequence that has a higher protein (peptide) expression rate compared to the endogenous RBS sequence, but is not limited thereto.

[0093] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of the protein (peptide) can enhance the activity of the protein (peptide) by introducing modifications to the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide) through deletion, insertion, substitution, or a combination thereof, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has increased activity, but is not limited thereto. Specifically, substitution can be achieved by inserting polynucleotides into the chromosome through homologous recombination, but is not limited thereto.

[0094] 6) The introduction of exogenous polynucleotides exhibiting the activity of the protein (peptide) can be achieved by introducing an exogenous polynucleotide encoding a protein (peptide) exhibiting the same / similar activity as the protein (peptide) into a host cell (microorganism). The exogenous polynucleotide is not limited by its source or sequence, as long as it exhibits the same / similar activity as the protein (peptide). Introduction can be carried out using transformation methods known in the art, appropriately selected by those skilled in the art, and the expression of the introduced polynucleotide in the host cell can produce the protein (peptide), thereby increasing its activity.

[0095] 7) Codon optimization of the polynucleotide encoding the protein (peptide) can be achieved by codon optimization of endogenous polynucleotides to increase transcription or translation in the host cell (microorganism), or by optimizing the codons of exogenous polynucleotides to achieve optimized transcription and translation in the host cell.

[0096] 8) By analyzing the tertiary structure of the protein (peptide), the selection and modification or chemical modification of the exposed region of the protein (peptide) can be achieved by, for example, comparing the sequence information of the protein (peptide) to be analyzed with a database storing the sequence information of known proteins to identify template protein candidates based on sequence similarity, and confirming the structure based on this information, thereby selecting the exposed site to be modified or chemically modified, and transforming or modifying the site.

[0097] 9) Regulation of the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to specific intracellular organelles or specific intracellular spaces. For example, this can be achieved by adding or removing a leader sequence that functions in targeting the protein (peptide), thereby targeting the periplasm or cytoplasm, but is not limited thereto.

[0098] This enhancement of protein (peptide) activity can refer to, but is not limited to, an increase in the activity or concentration of the protein (peptide) as expressed in the wild-type strain or host cell (microorganism) before modification, or an increase in the amount of product produced by the protein (peptide).

[0099] Modification of some or all of the polynucleotides in the host cell (microorganism) of this disclosure can be induced by: (a) using homologous recombination methods that use vectors for chromosome insertion or use engineered nucleases for genome editing (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto.

[0100] nourish

[0101] As used herein, the term "culture" refers to the cultivation of microorganisms under appropriately controlled environmental conditions. The cultivation process can be carried out in suitable culture media and under suitable cultivation conditions known in the art. Those skilled in the art can readily adapt and use such a cultivation process depending on the selected microorganisms. Specifically, the cultivation can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto.

[0102] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for the cultivation of microorganisms as its main components, and that the culture medium provides nutrients, growth factors, etc., including water, which is essential for survival and development. Specifically, any culture medium and culture conditions can be used to cultivate the microorganisms of this disclosure without particular limitation, as long as the culture medium is used for the general culture of microorganisms. For example, the microorganisms of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc. For example, culture media for Corynebacterium spp. can be found in the literature [Manual of Methods for General Bacteriology, American Society for Bacteriology (Washington DC, USA, 1981)].

[0103] In this disclosure, carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; or amino acids such as glutamic acid, methionine, and lysine. Additionally, natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and various other carbon sources can be used without limitation in appropriate amounts. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0104] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, or organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0105] Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts can be used as phosphorus sources. Sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., can be used as inorganic compounds. In addition, amino acids, vitamins, and / or suitable precursors can be included. These components or precursors can be added to the culture medium in batches or continuously. However, the culture medium is not limited to these.

[0106] During the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid in an appropriate manner. During cultivation, antifoaming agents such as polyethylene glycol fatty acids can be used to suppress foaming. To maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected. To maintain an anaerobic or slightly aerobic state in the culture medium, no gas injection is necessary, or nitrogen, hydrogen, or carbon dioxide gas can be injected. However, the cultivation conditions are not limited to these.

[0107] In the cultivation of this disclosure, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 to 160 hours, but the cultivation conditions are not limited to these.

[0108] As used herein, the term "culture" refers to a culture medium, a concentrated culture medium, a dried product of a culture medium, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate obtained by culturing a specific microorganism in a culture medium. A culture medium is a solution containing a specific microorganism, while a culture filtrate is a solution substantially free of the specific microorganism (where "substantially" means that the specific microorganism has been excluded by means of filtration, etc., but does not mean that the microorganism is completely absent from the filtrate). The form of a culture is not limited, and in one instance, it may be in the form of a liquid, an emulsion, or a solid.

[0109] As used herein, the term "fermentation" refers to the process by which microorganisms use their enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction proceed using similar processes. However, during decomposition, fermentation produces useful substances, while putrefaction produces foul odors or harmful substances.

[0110] In this disclosure, the method for obtaining fermentation products from microorganisms is not particularly limited and can be obtained according to methods conventionally used in related or similar technical fields.

[0111] As used herein, the term "fermentation product" includes not only the fermentation material itself, but also all types of substances containing fermentation products produced by microorganisms, including substances containing fermenting microorganisms, cultures produced by fermenting microorganisms, fermentation products of cultures, concentrated fermentation products, dried products of fermentation products, filtrates of fermentation products, filtrates of concentrated fermentation products, dried products of filtrates of fermentation products, extracts of fermentation products, or dilute solutions of fermentation products.

[0112] Specific Implementation Methods of This Disclosure

[0113] The specific implementation of this disclosure will be described in more detail below.

[0114] One aspect of this disclosure provides a Corynebacterium microorganism with L-tryptophan production capacity, wherein a ribo-phosphoro-pyrophosphate kinase or a polynucleotide encoding the ribo-phosphoro-phosphorokinase derived from Bacillus subtilis has been introduced into the microorganism.

[0115] As used herein, the term "ribose-phosphopyrophosphate kinase (PRSA)" refers to an enzyme that converts ribose 5-phosphate to phosphoribosyl pyrophosphate. The ribose-phosphopyrophosphate kinase of this disclosure may be used interchangeably with "PRSA".

[0116] Specifically, the ribo-phospho-pyrokinase disclosed herein may be a protein encoded by the prsA gene that has ribo-phospho-pyrokinase activity, but is not particularly limited thereto, as long as it exhibits activity corresponding to ribo-phospho-pyrokinase activity. Ribo-phospho-pyrokinases encoded by the prsA gene are known in the art, and the amino acid and polynucleotide sequences of ribo-phospho-pyrokinases can be obtained from publicly available databases, examples of which include, but are not limited to, NCBI's GenBank.

[0117] In one example, a ribo-phospho-pyrokinase derived from Bacillus subtilis may contain the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 60% homology or identity with it, but is not limited thereto, as long as it retains ribo-phospho-pyrokinase activity. Furthermore, it is apparent that even if a protein contains the amino acid sequence of SEQ ID NO:1, wherein a portion of that sequence is deleted, modified, substituted, or added, the protein may fall within the scope of this disclosure as long as it exhibits efficacy corresponding to that of ribo-phospho-pyrokinase. Proteins exhibiting equivalent efficacy to ribo-phospho-pyrokinase, having, containing, consisting of, or substantially consisting of the following amino acid sequences, may be included in ribo-phospho-pyrokinase, wherein the amino acid sequences have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO:1.

[0118] Furthermore, a sequence encoding a polynucleotide sequence of ribo-phospho-pyrokinase derived from Bacillus subtilis can be obtained, for example, based on codon information known in the art, wherein the ribo-phospho-pyrokinase has the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 60% homology or identity with it. In one example, the ribo-phospho-pyrokinase may be encoded by a polynucleotide having, comprising, consisting of, or substantially consisting of the sequence of SEQ ID NO: 2 or a base sequence having at least 60%, 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% homology or identity with the sequence of SEQ ID NO: 2, but is not limited thereto. The base sequence of SEQ ID NO: 2 can be obtained from known databases, such as NCBI's GenBank, but is not limited thereto.

[0119] In this disclosure, the term “polynucleotide (gene) containing the base sequence of SEQ ID NO: 2” may be used interchangeably with the terms “polynucleotide (gene) having the base sequence of SEQ ID NO: 2”, “polynucleotide (gene) composed of the base sequence of SEQ ID NO: 2”, or “prsA”.

[0120] Considering codon degeneracy or preferred codons in organisms to express the ribo-phospho-pyrokinase of this disclosure, the polynucleotides of this disclosure can accept various modifications in the coding region without altering the amino acid sequence of the ribo-phospho-pyrokinase. Therefore, it is apparent that polynucleotides that can be codonally degenerate into polypeptides consisting of the amino acid sequence of the ribo-phospho-pyrokinase of this disclosure, or polypeptides having at least 60% homology or identity with it, can also be included in the polynucleotides of this disclosure. For example, the polynucleotides of this disclosure can be the sequence of SEQ ID NO: 2, or its degenerate sequence.

[0121] In another instance, the polynucleotide of this disclosure may have or comprise a base sequence having at least 60%, 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% homology or identity with the sequence of SEQ ID NO: 2, or may consist of or substantially consist of a base sequence having at least 60%, 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% homology or identity with the sequence of SEQ ID NO: 2, but is not limited thereto.

[0122] Furthermore, the polynucleotides of this disclosure may include, without limitation, probes prepared from known gene sequences, for example, any sequence encoding the ribo-phospho-pyrokinase of this disclosure by hybridization under stringent conditions with a sequence that is fully or partially complementary to the polynucleotide sequence of this disclosure.

[0123] The Corynebacterium species disclosed herein exhibit L-tryptophan production capacity.

[0124] For the purposes of this disclosure, the microorganisms of this disclosure may include any microorganism capable of producing desired L-tryptophan, wherein ribose-phospho-pyrokinase or a polynucleotide encoding it is introduced. For example, the microorganisms of this disclosure may be characterized by an increased L-tryptophan production capacity due to the introduction of ribose-phospho-pyrokinase or a polynucleotide encoding it, and such microorganisms may be genetically modified or recombinant microorganisms, but are not limited thereto. Specifically, recombinant strains with increased L-tryptophan production capacity may be microorganisms with increased L-tryptophan production capacity compared to natural wild-type microorganisms, unmodified microorganisms with inherent ribose-phospho-pyrokinase activity, or unmodified microorganisms without inherent ribose-phospho-pyrokinase activity, but are not limited thereto.

[0125] In one instance, a microorganism capable of producing L-tryptophan refers to a prokaryotic or eukaryotic microorganism capable of producing L-tryptophan in a living organism, and may include any microorganism exhibiting increased L-tryptophan production capacity, or any microorganism conferred L-tryptophan production capacity by introducing the ribo-phospho-pyrophosphate kinase activity of this disclosure into microorganisms that have or do not possess inherent L-tryptophan production capacity. L-tryptophan production capacity can be conferred or enhanced through strain modification.

[0126] The microorganisms disclosed herein may include any microorganism into which ribose-phospho-pyrokinase or a polynucleotide encoding the ribose-phospho-pyrokinase has been introduced using various known methods.

[0127] In one instance, the recombinant microorganisms with L-tryptophan production capabilities disclosed herein may include any microorganism capable of producing L-tryptophan via vector transformation, wherein the vector introduces an exogenous gene encoding the ribo-phospho-pyrokinase of this disclosure (particularly ribo-phospho-pyrokinase derived from Bacillus subtilis).

[0128] For example, a microorganism that produces L-tryptophan may be a microorganism that introduces a protein encoding an amino acid sequence containing SEQ ID NO: 1 or a protein containing an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1.

[0129] For example, a microorganism that produces L-tryptophan can be a microorganism that introduces a polynucleotide capable of encoding a protein containing an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 1, or a polynucleotide containing the base sequence of SEQ ID NO: 2, or a base sequence having at least 60%, 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% homology or identity with the base sequence of SEQ ID NO: 2.

[0130] In one instance, the microorganism with increased L-tryptophan production capacity disclosed herein may be, but is not limited to, a microorganism with increased L-tryptophan production capacity compared to an unmodified microorganism. In one instance, the unmodified microorganism used as a reference strain for comparing increased L-tryptophan production capacity may be strain CM05-9157, but is not limited to.

[0131] In one instance, a microorganism with increased L-tryptophan production capacity may exhibit an increase of at least about 1% compared to the L-tryptophan production capacity of its parental microorganism (parental strain) or unmodified microorganism, specifically, at least about 1%, at least about 2.5%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, or at least about 21% (with no particular upper limit, e.g., up to about 200%, up to about 150%, up to about 100%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, or up to about 25%). However, the L-tryptophan production capacity of a microorganism is not limited to this, as long as it exhibits a positive increase compared to the production capacity of its parental microorganism (parental strain) or unmodified microorganism. In another instance, a recombinant microorganism with increased L-tryptophan production capacity may have an increase of at least about 1.1 times, at least about 1.15 times, at least about 1.16 times, at least about 1.17 times, at least about 1.18 times, at least about 1.19 times, at least about 1.2 times, or at least about 1.21 times compared to the parent microorganism before modification (parental strain) or the unmodified microorganism (there is no particular upper limit, e.g., up to about 10 times, up to about 5 times, up to about 3 times, up to about 2 times, up to about 1.5 times, up to about 1.4 times, up to about 1.3 times, or up to about 1.25 times), but is not limited thereto.

[0132] In one instance, the microorganisms with L-tryptophan production capacity disclosed herein may be prokaryotic or eukaryotic cells, specifically prokaryotic cells. Prokaryotic cells may include, for example, microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacterium*, *Pseudomonas*, *Leptospira*, *Salmonella*, *Brevibacteria*, *Hyphomonas*, *Chromobacterium*, or *Nocardia*, or microorganisms belonging to the fungi or yeasts, but are not limited thereto. Specifically, it can be a microorganism of the genus Escherichia, the genus Corynebacterium, the genus Leptospira, or yeast. More specifically, it can be a microorganism of the genus Corynebacterium.

[0133] In any of the aforementioned specific embodiments, the microorganisms disclosed herein may be Corynebacterium genus microorganisms.

[0134] In one embodiment of this disclosure, the microorganisms disclosed may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms disclosed herein may be Corynebacterium genus microorganisms, more specifically Corynebacterium glutamicum, but are not limited thereto.

[0135] Specifically, the microorganisms disclosed herein may be Corynebacterium genus microorganisms, more specifically Corynebacterium glutamicum, but are not limited thereto.

[0136] Meanwhile, the Corynebacterium genus microorganisms with L-tryptophan production capacity disclosed herein may include any naturally wild-type microorganism, Corynebacterium genus microorganisms with increased L-tryptophan production capacity by enhancing or weakening the expression of genes related to the L-tryptophan production mechanism, or Corynebacterium genus microorganisms with increased L-tryptophan production capacity by introducing or enhancing exogenous genes.

[0137] Another aspect of this disclosure provides a method for producing L-tryptophan, comprising culturing a Corynebacterium microorganism of the present disclosure capable of producing L-tryptophan in a culture medium, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding therethe.

[0138] In the methods disclosed herein, the cultivation of microorganisms can be carried out using any culture conditions and methods known in the art. Those skilled in the art can readily adapt and use this culture process according to the selected strain.

[0139] L-tryptophan produced through the culture of this disclosure can be secreted into the culture medium or retained inside the cell.

[0140] In one specific embodiment, the method for producing L-tryptophan disclosed herein may further include, for example, preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the microorganisms, or a combination thereof (in any order) prior to culturing.

[0141] In one specific embodiment, the method may further include recovering the desired substance, particularly L-tryptophan, from cultured microorganisms, microbial cultures, microbial fermentation products, or culture media. A recovery step may also be included after culturing.

[0142] Recovery can be achieved by collecting the desired L-tryptophan using suitable methods known in the art, according to the methods for culturing microorganisms disclosed herein (e.g., batch, continuous, or fed-batch culture). For example, recovery may include centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), HPLC, or combinations thereof. Using suitable methods known in the art, the desired substance, particularly L-tryptophan, can be recovered from the culture medium or microorganisms.

[0143] Furthermore, the method for producing L-tryptophan disclosed herein may further include purification. Purification can be performed using suitable methods known in the art. In one instance, when the method for producing L-tryptophan disclosed herein includes both recovery and purification, recovery and purification may be performed continuously, discontinuously, in any order, simultaneously, or as an integrated step, but are not limited thereto.

[0144] In the methods disclosed herein, the ribo-phospho-pyrokinase, the introduction, L-tryptophan, etc., are as described above.

[0145] Another aspect of this disclosure provides a composition for producing L-tryptophan, comprising: a Corynebacterium microorganism having L-tryptophan production capability, said microorganism having incorporated a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding thereus; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more of these.

[0146] The compositions disclosed herein may also contain any suitable excipients commonly used in compositions for the production of L-tryptophan. Such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0147] In one specific embodiment, the composition disclosed herein may comprise each component in a microbially effective amount or in an amount suitably present in the composition used for production.

[0148] In the compositions disclosed herein, the ribo-phospho-pyrokinase, the introduction, L-tryptophan, etc., are as described above.

[0149] Another aspect of this disclosure provides the use of a Corynebacterium microorganism with L-tryptophan production capability for the production of L-tryptophan, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the ribo-phospho-pyrophosphate kinase.

[0150] In the uses disclosed herein, the ribo-phospho-pyrokinase, the introduction, L-tryptophan, etc., are as described above.

[0151] [Method for implementing the present invention]

[0152] This disclosure will be described in detail by way of examples. However, these examples are given for illustrative purposes only, and the scope of this disclosure is not intended to be limited by these examples. Furthermore, any technical descriptions lacking in this disclosure can be fully understood and readily practiced by those skilled in the art to which this disclosure pertains or in related fields.

[0153] Example 1. Identification and selection of ribose-phospho-pyrokinase (prsA) gene

[0154] To identify candidate genes and organisms possessing an enzyme believed to encode the conversion of ribose 5-phosphate to phosphoribosyl pyrophosphate, a PSI-BLAST search was performed using the amino acid sequence of prsA (a ribose-phosphopyrophosphate kinase derived from Corynebacterium glutamicum) as the query sequence, based on the NCBI and KEGG databases. As a result, considering the biosafety level suitable for the production strain and the feasibility of protecting the organism, three organisms were selected, as shown in Table 1 below.

[0155] [Table 1]

[0156]

[0157] Example 2. Preparation of microorganisms for producing L-tryptophan, wherein exogenous ribo-phospho-pyrokinase was introduced into the microorganisms.

[0158] Example 2-1. Construction of plasmids for gene insertion

[0159] To insert the gene into the Corynebacterium chromosome, plasmid pDCM2 (Korean Patent No. 10-2278000) was used as the parental vector, and to enhance the activity of ribose-phospho-pyrophosphate kinase, a plasmid for additional insertion of the prsA gene was constructed using the Pcj7 promoter (Korean Patent No. 10-0620092).

[0160] Specifically, chromosomal DNA from wild-type Corynebacterium glutamicum ATCC13869 was used as a template. The upstream region of the chromosome undergoing homologous recombination was amplified using primer pairs SEQ ID NO: 9 and 10, and the downstream region was amplified using primer pairs SEQ ID NO: 11 and 12. PCR was then performed to obtain each gene fragment. PCR was performed using Solg™ Pfu-X DNA polymerase (SolGent Co.) under the following conditions: denaturation at 95°C for 4 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 50 seconds, and a final extension at 72°C for 5 minutes. The primer sequences used are shown in Table 2 below.

[0161] [Table 2]

[0162]

[0163] The recombinant plasmid was obtained by cloning upstream and downstream fragments of the chromosome region amplified by the above PCR and subjected to homologous recombination using the Gibson assembly method (DG Gibson et al., Nature Methods, Vol. 6, No. 5, May 2009; NEBuilder HiFi DNA Assembly Master Mix), along with the chromosome transformation vector pDCM2 (Korean Patent No. 10-2278000) digested with restriction enzymes EcoRI and SalI. This plasmid was named pDCM2-ΔTn.

[0164] Example 2-2. Preparation of Corynebacterium microorganisms incorporating ribo-phospho-pyrokinase derived from Bacillus subtilis

[0165] First, to obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7662943 B2) as a template and primers SEQ ID NO: 13 and 14. The polymerase used was Solg™ Pfu-X DNA polymerase (SolGentCo.), and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of: denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The primer sequences used are shown in Table 3 below.

[0166] [Table 3]

[0167]

[0168] The gene encoding ribo-phospho-pyrokinase from Bacillus subtilis selected in Example 1 has the amino acid sequence of SEQ ID NO: 1. Information on the gene encoding ribo-phospho-pyrokinase and its surrounding base sequence (NC_000964.3, SEQ ID NO: 2) was obtained from NIH GenBank. Based on the obtained base sequence, primers were synthesized to insert the gene from Bacillus subtilis into the genomic DNA of Corynebacterium glutamicum. The ribo-phospho-pyrokinase gene (SEQ ID NO: 2) from Bacillus subtilis was synthesized using the gene synthesis service of Bionics Co., Ltd., and amplified by PCR using primer pairs of SEQ ID NO: 15 and 16. The polymerase used was Solg™ Pfu-X DNA polymerase, and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes. The primer sequences used are shown in Table 4 below.

[0169] [Table 4]

[0170]

[0171] Next, the Pcj7 promoter region amplified as described above, the gene fragment derived from Bacillus subtilis, and the chromosome transformation vector pDCM2-ΔTn prepared in Example 2-1 and digested with the restriction enzyme ScaI were cloned using the Gibson assembly method (DG Gibson et al., Nature Methods, Vol. 6, No. 5, May 2009; NEBuilder HiFi DNA Assembly Master Mix) to obtain the recombinant plasmid, which was named pDCM2-ΔTn::Pcj7-prsA(B.su). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment at a calculated molar ratio and incubating the mixture at 50°C for 1 hour. The constructed pDCM2-ΔTn::Pcj7-prsA(B.su) vector was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541–545). After a second crossover, a strain was obtained in which one copy of the Pcj7_prsA(B.su) gene was inserted between transposon genes on the chromosome. The strain was identified by PCR and genome sequencing using primers SEQ ID NO: 17 and 18, which amplified the outer regions of the homologous recombination sites upstream and downstream of the gene insertion locus, respectively. The primer sequences used are shown in Table 5 below.

[0172] [Table 5]

[0173]

[0174] The strain obtained by the above method was named CM05-9157△Tn::Pcj7_prsA(b.su).

[0175] Examples 2-3. Preparation of Corynebacterium microorganisms incorporating ribo-phospho-pyrokinase derived from Mycobacterium smegmatis

[0176] First, to obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7662943 B2) as a template and primers from SEQ ID NO: 13 in Table 3 and SEQ ID NO: 19 in Table 6. The polymerase used was Solg™ Pfu-X DNA polymerase (SolGent Co.), and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of the following: denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.

[0177] [Table 6]

[0178]

[0179] The ribo-phospho-pyrokinase gene selected in Example 1 from *Mycobacterium smegmatis* has the amino acid sequence SEQ ID NO: 3. Information about the corresponding gene and surrounding base sequence (NZ_CP054795.1, SEQ ID NO: 4) was obtained from NIH GenBank. Based on the obtained base sequence, primers were synthesized to insert the gene from *Mycobacterium smegmatis* into the genomic DNA of *Corynebacterium glutamicum*. The ribo-phospho-pyrokinase gene (SEQ ID NO: 4) from *Mycobacterium smegmatis* was synthesized using the gene synthesis service of Bionics Co., Ltd., and amplified by PCR using primer pairs SEQ ID NO: 20 and 21 in the same manner as in Examples 2-2. The primer sequences used are shown in Table 7 below.

[0180] [Table 7]

[0181]

[0182] Next, the Pcj7 promoter region amplified as described above, the gene fragment derived from Mycobacterium smegmatis, and the chromosome transformation vector pDCM2-ΔTn prepared in Example 2-1, digested with the restriction enzyme ScaI, were cloned using the Gibson assembly method to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-prsA(m.sm). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment at a calculated molar ratio and incubating the mixture at 50°C for 1 hour. The constructed pDCM2-ΔTn::Pcj7-prsA(m.sm) vector was transformed into tryptophan-producing strain CM05-9157 (Korean Patent Registration No. 10-2278000) by electroporation, and after a second exchange process, a strain was obtained in which one copy of the Pcj7_prsA(m.sm) gene was inserted between transposon genes on the chromosome. The strain was identified by PCR and genome sequencing using primers in Table 5, which amplified the outer regions of the upstream and downstream homologous recombination sites of the gene insertion locus, respectively.

[0183] The strain obtained by the above method was named CM05-9157△Tn::Pcj7-prsA(m.sm).

[0184] Examples 2-4. Preparation of Corynebacterium microorganisms incorporating ribo-phospho-pyrokinase derived from Escherichia coli

[0185] First, to obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7662943 B2) as a template and primers from SEQ ID NO: 13 in Table 3 and SEQ ID NO: 22 in Table 8. The polymerase used was Solg™ Pfu-X DNA polymerase (SolGent Co.), and PCR amplification was performed under the following conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of the following: denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.

[0186] [Table 8]

[0187]

[0188] The ribo-phospho-pyrokinase selected in Example 1, derived from *E. coli*, has the amino acid sequence SEQ ID NO: 5. Information about the corresponding gene and surrounding base sequence (NC_007779.1, SEQ ID NO: 6) was obtained from NIH GenBank. Based on the obtained base sequence, primers were synthesized to insert the *E. coli*-derived gene into the genomic DNA of *Corynebacterium glutamicum*.

[0189] The ribose-phospho-pyrokinase gene (SEQ ID NO: 6) derived from *E. coli* was synthesized using the gene synthesis service of Bionics Co., Ltd., and amplified by PCR using primer pairs SEQ ID NO: 23 and 24 in the same manner as in Examples 2-2. The primer sequences used are shown in Table 9 below.

[0190] [Table 9]

[0191]

[0192] Next, the amplified Pcj7 promoter region, the gene fragment derived from *E. coli*, and the chromosome transformation vector pDCM2-ΔTn prepared in Example 2-1 and digested with the restriction enzyme ScaI were cloned using the Gibson assembly method to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-prsA(e.co). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment at a calculated molar ratio and incubating the mixture at 50°C for 1 hour. The constructed pDCM2-ΔTn::Pcj7-prsA(e.co) vector was transformed into tryptophan-producing strain CM05-9157 (Korean Patent Registration No. 10-2278000) by electroporation, and after a second exchange process, a strain was obtained in which one copy of the Pcj7_prsA(e.co) gene was inserted between transposon genes on the chromosome. The strain was identified by PCR and genome sequencing using primers of SEQ ID NO: 17 and 18 in Table 5, wherein the PCR was able to amplify the outer regions of the upstream and downstream homologous recombination sites of the gene insertion locus, respectively.

[0193] The strain obtained by the above method was named CM05-9157△Tn::Pcj7-prsA(e.co).

[0194] Examples 2-5. Preparation of Corynebacterium species with further enhanced ribo-phospho-pyrokinase derived from Corynebacterium glutamicum.

[0195] First, in order to obtain the Pcj7 promoter, PCR was performed in the same manner as in Examples 2-2, using p117-cj7-gfp (US 7662943 B2) as a template and primers SEQ ID NO: 13 in Table 3 and SEQ ID NO: 25 in Table 10.

[0196] [Table 10]

[0197]

[0198] To further enhance ribophosphate pyrophosphate kinase by inserting the gene (NZ_CP016335.1, SEQ ID NO: 8) encoding ribophosphate pyrophosphate kinase (SEQ ID NO: 7) of wild-type Corynebacterium glutamicum ATCC13869 into the genomic DNA, PCR was performed using chromosomal DNA of wild-type Corynebacterium glutamicum ATCC 13869 strain as a template and primers of SEQ ID NO: 26 and 27 in the same manner as in Examples 2-2. The primer sequences used are shown in Table 11 below.

[0199] [Table 11]

[0200]

[0201] Next, the amplified Pcj7 promoter region, the gene fragment derived from Corynebacterium glutamicum, and the chromosome transformation vector pDCM2-ΔTn prepared in Example 2-1 and digested with the restriction enzyme ScaI were cloned using the Gibson assembly method to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-prsA(C.gl). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment at a calculated molar ratio and incubating the mixture at 50°C for 1 hour. The constructed pDCM2-ΔTn::Pcj7-prsA(C.gl) vector was transformed into tryptophan-producing strain CM05-9157 (Korean Patent Registration No. 10-2278000) by electroporation, and after a second exchange process, a strain was obtained in which one copy of the Pcj7-prsA(C.gl) gene was inserted between transposon genes on the chromosome. The strain was identified by PCR and genome sequencing using primers in Table 5, which amplified the outer regions of the upstream and downstream homologous recombination sites of the gene insertion locus, respectively.

[0202] The strain obtained by the above method was named CM05-9157△Tn::Pcj7_prsA(c.gl).

[0203] Example 3. Evaluation of L-tryptophan production capacity of Corynebacterium microorganisms with introduced exogenous ribose-phosphopyrokinase.

[0204] To evaluate the L-tryptophan production capacity of the parental strain CM05-9157 without the introduction of exogenous genes, and the production strains constructed in Examples 2-2, 2-3, 2-4, and 2-5 in which a copy of the ribose-phosphophosphoryl kinase gene was additionally enhanced (i.e., CM05-9157ΔTn::Pcj7_prsA(b.su), CM05-9157ΔTn::Pcj7_prsA(m.sm), CM05-9157ΔTn::Pcj7_prsA(e.co), and CM05-9157ΔTn::Pcj7_prsA(c.gl)), the strains were cultured using the following method.

[0205] Each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of seed culture medium and incubated at 30°C with shaking at 200 rpm for 20 hours. Then, 1 mL of the seed culture was inoculated into a 250 mL corner-baffled flask containing 25 mL of production culture medium and incubated at 30°C with shaking at 200 rpm for 24 hours. After incubation, the amount of L-tryptophan produced was measured by HPLC. The compositions of the seed and production media are shown below, and the L-tryptophan concentration in the culture medium for each test strain is shown in Table 12.

[0206] [Seed culture medium (pH 7.0)]

[0207] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4·7H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, and 2000 μg nicotinamide (per 1 L distilled water).

[0208] [Production medium (pH 7.0)]

[0209] 30 g glucose, 15 g (NH4)2SO4, 1.2 g MgSO4·7H2O, 1 g KH2PO4, 5 g yeast extract, 900 μg biotin, 4500 μg thiamine hydrochloride, 4500 μg calcium pantothenate, 30 μg CaCO3 (per 1 L distilled water).

[0210] [Table 12]

[0211]

[0212] As shown in Table 12, the amount of tryptophan produced by the CM05-9157ΔTn::Pcj7_prsA(c.gl) strain (in which the ribo-phospho-pyrophosphate kinase gene derived from Corynebacterium glutamicum was additionally enhanced) was 1.92 g / L, which was almost the same as the amount of tryptophan produced by the parent strain CM05-9157.

[0213] In contrast, the CM05-9157ΔTn::Pcj7_prsA(b.su) strain, which incorporated a gene from Bacillus subtilis, produced 2.32 g / L of L-tryptophan in shake-flask culture, indicating a fermentation yield increase of approximately 21% compared to the CM05-9157ΔTn::Pcj7_prsA(c.gl) strain.

[0214] Compared with the control strain CM05-9157, strains CM05-9157ΔTn::Pcj7_prsA(m.sm) and CM05-9157ΔTn::Pcj7_prsA(e.co) showed a slight decrease or only a relatively slight increase in tryptophan production.

[0215] These results indicate that, among the exogenous ribo-phosphoro-pyrophosphate kinase genes introduced into Corynebacterium microorganisms, only the introduction of the ribo-phosphoro-pyrophosphate kinase gene derived from Bacillus subtilis can specifically enhance L-tryptophan production.

[0216] As described above, those skilled in the art will understand that this disclosure may be implemented in other specific forms without departing from the technical spirit or essential characteristics of this disclosure. In this regard, it should be understood that the foregoing embodiments are illustrative in all respects and should not be construed as restrictive. The scope of this disclosure should be interpreted to include the meaning and scope of the appended claims rather than the specific embodiments, and all changes or variations derived from equivalent concepts fall within the scope of this disclosure.

[0217] [Sequence List]

[0218]

[0219]

[0220]

[0221]

Claims

1. A Corynebacterium microorganism with L-tryptophan production capacity, wherein the microorganism has been introduced with ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the phospho-pyrophosphate kinase.

2. The microorganism according to claim 1, wherein the ribo-phospho-pyrophosphate kinase comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity with it.

3. The microorganism according to claim 1, wherein the ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis is encoded by the prsA gene.

4. The microorganism according to claim 1, wherein the polynucleotide encoding ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis comprises the base sequence of SEQ ID NO:

2.

5. The microorganism according to claim 1, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

6. The microorganism according to any one of claims 1 to 5, wherein the microorganism exhibits increased L-tryptophan production capacity compared with unmodified microorganisms.

7. A method for producing L-tryptophan, comprising culturing a Corynebacterium genus microorganism capable of producing L-tryptophan in a culture medium, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the ribo-phospho-pyrophosphate kinase.

8. The method of claim 7, wherein the method further comprises recovering L-tryptophan from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, or said culture medium.

9. A composition for producing L-tryptophan, comprising: a Corynebacterium microorganism having L-tryptophan production capability, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the same; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more thereof.

10. The use of a Corynebacterium microorganism with L-tryptophan production capacity for the production of L-tryptophan, wherein the microorganism has been introduced with a ribo-phospho-pyrophosphate kinase derived from Bacillus subtilis or a polynucleotide encoding the ribo-phospho-pyrophosphate kinase.

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