L-alanine-producing microorganism and method for producing L-alanine using the same

Abstract

The present application relates to a Corynebacterium microorganism having L-alanine production ability into which alanine dehydrogenase activity has been introduced, a method for producing L-alanine, the method comprising a step of culturing the microorganism in a medium, a composition for producing L-alanine, the composition comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of at least two of them, and use of the microorganism for L-alanine production.

Description

[Technical Field]

[0001] The present application relates to a Corynebacterium microorganism having L-alanine production ability into which alanine dehydrogenase activity has been introduced, a method for producing L-alanine, the method comprising a step of culturing the microorganism in a medium, a composition for producing L-alanine, the composition comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of at least two of them, and use of the microorganism for L-alanine production. [Background technology]

[0002] L-alanine is an important amino acid with a wide range of applications in the fields of chemistry, food, and medicine. It is a sweet-tasting amino acid and is used in the food industry, particularly as a flavor enhancer and nutritional supplement. The global alanine market in 2020 was valued at over USD 250 million, with an annual production of approximately 500 tons (The Expresswire, 2020; Wendisch, 2014).

[0003] In this regard, chemical synthesis or enzymatic conversion is mainly used to produce L-alanine. Chemical synthesis is performed using a modified Strecker synthesis or Bucherer-Bergs method (Legnani et al., 2021), while enzymatic conversion is obtained from L-aspartic acid using ammonium fumarate (Takamatsu et al., 1982). Most industrial production is carried out via enzymatic conversion, but the substrate, L-aspartic acid, is produced from petroleum, and fumaric acid production is also dependent on petroleum, resulting in high costs and low yields when used as a substitute.

[0004] Therefore, as an alternative, microbial fermentation can provide an abundant carbon source at low cost. In nature, microorganisms produce L-alanine using aminotransferase or alanine dehydrogenase. However, the production of L-alanine using naturally occurring strains is limited by low productivity.

[0005] Therefore, there is still a need for research to effectively improve L-alanine productivity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,662,943 [Patent Document 2] U.S. Patent No. 10,584,338 [Patent Document 3] U.S. Patent No. 10,273,491 [Patent Document 4] Korean Patent Publication No. 10-2020-0136813 [Non-patent literature]

[0007] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-patent document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-patent document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-patent document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)

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[0008] The present inventors have confirmed that when alanine dehydrogenase activity is introduced into a Corynebacterium microorganism, the L-alanine-producing ability is improved compared to an unmodified microorganism, and have completed the present application. [Means for solving the problem]

[0009] One aspect of the present application provides a Corynebacterium microorganism having an ability to produce L-alanine, into which alanine dehydrogenase activity has been introduced.

[0010] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0011] In other embodiments, the alanine dehydrogenase may be encoded by the ald1, ald2, or alaD gene.

[0012] In yet other embodiments, the alanine dehydrogenase may comprise the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

[0013] In still another specific example, the gene encoding the alanine dehydrogenase may comprise the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0014] In any of the microorganisms of the aforementioned embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0015] In any of the specific examples of the microorganisms described above, the Corynebacterium microorganism may have improved L-alanine production ability compared to an unmodified microorganism.

[0016] Another aspect of the present application provides a method for producing L-alanine, comprising the step of culturing in a medium a Corynebacterium microorganism having an ability to produce L-alanine and into which alanine dehydrogenase activity has been introduced.

[0017] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0018] In other embodiments, the method may further comprise the step of recovering a target substance from the cultured microorganism, a culture of the microorganism, a fermentation product of the microorganism, or the culture medium.

[0019] Yet another aspect of the present application provides a composition for producing L-alanine, comprising a Corynebacterium microorganism having L-alanine production ability and into which alanine dehydrogenase activity has been introduced, a culture of the microorganism, a fermentation product of the microorganism, or a combination of at least two of them.

[0020] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0021] Yet another aspect of the present application provides use of a Corynebacterium microorganism into which alanine dehydrogenase activity has been introduced for the production of L-alanine.

[0022] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari. [Effects of the Invention]

[0023] The Corynebacterium microorganism having L-alanine-producing ability and into which the alanine dehydrogenase activity of the present invention has been introduced can produce L-alanine in high yield, and is therefore useful for the industrial production of L-alanine. DETAILED DESCRIPTION OF THE INVENTION

[0024] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the following specific descriptions.

[0025] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0026] As used in the specification and claims of this application, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated. Unless otherwise indicated, singular terms include plurals and plural terms include the singular. As used in the specification and claims of this application, "or" is used in its sense including "and / or" unless otherwise indicated.

[0027] In this application, the word "about" is used before a specific numerical value. In this application, "about" includes not only the exact numerical value following the term "about," but also a range that is approximately that numerical value or close to that numerical value. Considering the context in which the numerical value is used, it can be determined whether the numerical value is close to or approximately the specific numerical value referred to. As an example, "about" indicates a range of -10% to +10% of the specified numerical value. As another example, "about" indicates a range of -5% to +5% of the specified numerical value. However, the present invention is not limited to these examples.

[0028] In this application, terms such as "first, second, third," "i), ii), iii), "(a), (b), (c), (d)," etc. are used to distinguish between similar components and do not imply continuity or sequential order. For example, when the terms are used with respect to steps in a method, use, or analysis, the steps may be performed simultaneously or separated by intervals of a few seconds, minutes, hours, days, or months.

[0029] In this application, "consisting essentially of" means that unspecified components are present when the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0030] In this application, "consisting of" means that the percentages of the specified components total 100%. Components or features referred to as "consisting of" are essential or mandatory. In one embodiment, other optional or non-essential components are excluded from the components or features referred to as "consisting of."

[0031] In this application, "comprising" means the presence of the feature, step, or component referred to by the term, but does not exclude the presence or addition of one or more features, steps, or components. In this application, a component or feature referred to as "comprising" is essential or mandatory, but in an embodiment, the component or feature may further include other optional or non-essential components or features.

[0032] One aspect of the present application provides a Corynebacterium microorganism having an ability to produce L-alanine, into which alanine dehydrogenase activity has been introduced.

[0033] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0034] The alanine dehydrogenase activity is defined as, but is not limited to, improving the L-alanine production ability of a microorganism into which the alanine dehydrogenase activity of the present application has been introduced, compared to the production ability of a natural wild-type microorganism or an unmodified microorganism (e.g., a strain before the alanine dehydrogenase activity of the present application has been introduced).

[0035] For example, the activity of the alanine dehydrogenase can be determined by measuring the L-alanine productivity or yield, but is not limited thereto.

[0036] In the present application, "alanine dehydrogenase" refers to an oxidoreductase that catalyzes the reversible conversion of pyruvate to L-alanine with the coenzymes NAD+ / NADH.

[0037] Specifically, the alanine dehydrogenase of the present application is a protein having alanine dehydrogenase activity encoded by the ald1, ald2, or alaD gene, but the type is not particularly limited as long as it has activity equivalent to that of alanine dehydrogenase and improves L-alanine production ability when introduced into a Corynebacterium microorganism. The alanine dehydrogenases encoded by the ald1, ald2, or alaD gene are known in the art, and the amino acid and polynucleotide sequences of the alanine dehydrogenases can be obtained from publicly known databases, such as, but not limited to, GenBank at NCBI.

[0038] In the present application, the alanine dehydrogenase is, but is not limited to, alanine dehydrogenase derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari.

[0039] The alanine dehydrogenase derived from Bacillus licheniformis presented as an example in the present application is a protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, the alanine dehydrogenase derived from Bacillus amyloliquefaciens is a protein comprising the amino acid sequence of SEQ ID NO: 5, and the alanine dehydrogenase derived from Laceyella sacchari is a protein comprising the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0040] For example, in the present application, a protein having alanine dehydrogenase activity is a protein comprising the amino acid sequence of alanine dehydrogenase derived from Bacillus licheniformis (SEQ ID NO: 1 or SEQ ID NO: 3), the amino acid sequence of alanine dehydrogenase derived from Bacillus amyloliquefaciens (SEQ ID NO: 5), or the amino acid sequence of alanine dehydrogenase derived from Laceyella sacchari (SEQ ID NO: 7), but is not limited to these.

[0041] For example, the alanine dehydrogenase may comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or an amino acid sequence having 60% or more homology or identity thereto, but is not limited to these, as long as it has alanine dehydrogenase activity. Specifically, the polypeptide having alanine dehydrogenase activity may have the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or may comprise, consist of, or essentially consist of the amino acid sequence.

[0042] In this application, when an amino acid sequence is described as a polypeptide "comprising" an amino acid sequence represented by a particular SEQ ID NO, a polypeptide "consisting of" an amino acid sequence represented by a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence represented by a particular SEQ ID NO, it goes without saying that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the application, so long as they have the same or equivalent activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions thereof are not excluded, and it goes without saying that proteins having such sequence additions or mutations are also included in the application, so long as they have the same or equivalent activity as a protein consisting of the amino acid sequence of the SEQ ID NO.

[0043] For example, the variant polypeptide of the present application may have an addition of a sequence that does not alter the function of the variant polypeptide, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or internally of the amino acid sequence. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer. The polypeptide may also be linked to another sequence or linker to allow identification, purification, or synthesis of the polypeptide.

[0044] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature 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. Other examples include electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids. Valine, leucine, and isoleucine are classified as branched-chain amino acids.As another example, the 20 amino acids are classified by size and divided into five groups in order of decreasing relative volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. Conservative substitutions are typically not limited to these groups. Conservative substitutions typically have little or no effect on the activity of a polypeptide.

[0045] The alanine dehydrogenase-encoding nucleotide sequence may also be a nucleotide sequence encoding a protein that exhibits alanine dehydrogenase activity and, when introduced into a Corynebacterium microorganism, improves L-alanine production, such as, but not limited to, alanine dehydrogenase derived from Bacillus licheniformis (SEQ ID NO: 1 or SEQ ID NO: 3), alanine dehydrogenase derived from Bacillus amyloliquefaciens (SEQ ID NO: 5), or alanine dehydrogenase derived from Laceyella sacchari (SEQ ID NO: 7).

[0046] For example, an alanine dehydrogenase having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 may be encoded by a polynucleotide that has, contains, consists of, or is substantially composed of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. The nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 can be obtained from a publicly known database, such as, but not limited to, GenBank of NCBI.

[0047] In the present application, a gene comprising the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 is used interchangeably with a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, a gene or polynucleotide having the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, and a gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

[0048] The polynucleotide of the present application can be modified in various ways in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the alanine dehydrogenase of the present application is to be expressed, as long as the amino acid sequence of the alanine dehydrogenase of the present application is not changed. Therefore, it goes without saying that the polynucleotide of the present application also includes a polynucleotide that, due to codon degeneracy, is translated into a polypeptide consisting of the amino acid sequence of the alanine dehydrogenase of the present application, or a polypeptide having homology or identity thereto. For example, the polynucleotide of the present application may be SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a degenerated sequence thereof.

[0049] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application, and encodes the alanine dehydrogenase of the present application.

[0050] As used herein, "homology" or "identity" refers to the degree to which two given amino acid or nucleotide sequences are related, expressed as a percentage. Homology and identity are often used interchangeably.

[0051] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially, homologous or identical sequences will generally hybridize to at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or length under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having codons commonly used in polynucleotides or codons that take into account codon degeneracy.

[0052] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 1. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0053] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in, for example, Non-Patent Document 8. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) unitary matrices (identity takes a value of 1 and non-identity takes a value of 0), a PAM Matrix (see the disclosure in Non-Patent Document 9), a weighted comparison matrix from Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0054] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate defined hybridization conditions are within the skill of the art and are determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12), but are not limited to these.

[0055] In this application, "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Document 13). For example, conditions include those under which polynucleotides with high homology or identity, e.g., polynucleotides having a homology or identity of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, while polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, i.e., 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.

[0056] Such hybridization requires that the two nucleotides have complementary sequences, even though, depending on the stringency of the hybridization, mismatches between bases are possible.

[0057] The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar base sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0058] For example, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0059] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0060] In this application, "L-alanine" refers to an L-amino acid that is one of the essential amino acids and has the chemical formula HO2CCH(NH2)CH3.

[0061] In this application, "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism" and "strain" are used interchangeably and are used interchangeably.

[0062] For example, the microorganism of the present application is a microorganism (e.g., a recombinant strain) into which alanine dehydrogenase activity has been introduced, but is not limited thereto.

[0063] In the present application, the term "microorganism capable of producing L-alanine" refers to a prokaryotic or eukaryotic microbial strain that produces L-alanine in its organism, and includes microorganisms in which the ability to produce L-alanine has been imparted to a parent strain that does not have the ability to produce L-alanine, and microorganisms that have the ability to produce L-alanine endogenously. The ability to produce L-alanine can be imparted or improved by breeding.

[0064] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to wild-type or naturally occurring strains themselves, or strains before their characteristics are changed due to genetic mutations caused by natural or artificial factors. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "unmodified strain," "unmutated microorganism," "parent strain before mutation," "wild-type microorganism," "reference microorganism," or "reference microorganism." In the present application, "unmodified microorganism" refers to a strain into which the alanine dehydrogenase activity of the present application has not been introduced or before the alanine dehydrogenase activity has been introduced, but is not limited thereto. In the present application, "unmodified microorganism" refers to a microorganism that does not contain the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, but is not limited thereto.

[0065] For the purposes of this application, the microorganism of this application includes all microorganisms into which alanine dehydrogenase activity has been introduced and which produce the desired L-alanine. For example, the microorganism of this application is characterized by improved L-alanine production ability due to the introduction of alanine dehydrogenase activity, and is a genetically modified microorganism or a recombinant microorganism, but is not limited thereto. Specifically, the recombinant strain with improved L-alanine production ability is a microorganism with improved L-alanine production ability compared to a natural wild-type microorganism or an unmodified microorganism having endogenous alanine dehydrogenase activity, but is not limited thereto.

[0066] For example, a microorganism capable of producing L-alanine is a prokaryotic or eukaryotic microbial strain that produces L-alanine within the organism, and includes all microorganisms that have endogenous L-alanine-producing ability and microorganisms in which L-alanine-producing ability has been imparted to a parent strain that does not have L-alanine-producing ability by introducing alanine dehydrogenase activity into the microorganism in the present invention. L-alanine-producing ability can be imparted or improved by breeding.

[0067] The microorganisms of the present application include all microorganisms into which alanine dehydrogenase activity has been introduced by various known methods.

[0068] In the present application, "introduction" of an activity means expressing a gene that a microorganism does not originally have in the microorganism, thereby causing the activity of a specific protein to be manifested, or causing the activity of a polypeptide to be strengthened, increased, or improved compared to the endogenous activity of the protein or the activity before modification. For example, this may mean introducing a polynucleotide encoding a specific protein into a chromosome in a microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into a microorganism, causing the activity of the protein to be manifested.

[0069] The term "endogenous activity" refers to the activity of a polypeptide that a parent strain or an unmodified microorganism originally possessed before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is sometimes used interchangeably with "activity before modification."

[0070] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is interchangeable with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to the endogenous activity or the activity prior to modification. "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide compared to the activity and / or concentration (expression level) inherent in the parent strain or unmodified microorganism prior to transformation. For example, the "development of an activity not originally present" refers to, but is not limited to, "protein introduction."

[0071] The activity of a polypeptide being enhanced compared to the endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide is improved compared to the activity and / or concentration (expression level) that the strain or unmodified microorganism originally had before the transformation.

[0072] For example, the term "enhancement" means that a protein activity not originally present is manifested, or that the activity or concentration is increased by, generally, about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, or up to about 1000% or about 2000%, compared to the activity or concentration of the wild-type protein or the initial microbial strain, but is not limited to these. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any number that is equal to or in a similar range to the number following the term "about," but is not limited to these.

[0073] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0074] Various methods well known in the art can be applied to enhance the activity of the polypeptide, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, methods such as, but not limited to, conventional methods in molecular biology using genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 14 and 15).

[0075] Specifically, the enhancement of a polypeptide of the present application can be achieved by: 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide; 2) modifying the expression regulatory region of a gene on a chromosome that encodes the polypeptide (e.g., by generating a mutation in the expression regulatory region, substituting a sequence with a higher activity, or inserting a sequence with a higher activity); 3) modifying the nucleotide sequence encoding the start codon or 5'UTR region of a gene transcript that encodes the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance polypeptide activity); 6) introducing a foreign polypeptide that exhibits polypeptide activity or a foreign polynucleotide encoding it; 7) optimizing the codons of a polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed sites; or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0076] More specifically, the 1) intracellular copy number of a polynucleotide encoding a polypeptide may be increased by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the polynucleotide encoding the polypeptide may be introduced into a chromosome in the host cell at one or more copies. The introduction into a chromosome may be achieved by, but not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0077] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide (2) can be replaced with a sequence with stronger activity by, for example, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. Examples of the expression regulatory region include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, this can be achieved by replacing the original promoter with a strong promoter, but is not limited to this.

[0078] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 1), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 2), the O2 promoter (Patent Document 3), the tkt promoter, and the yccA promoter.

[0079] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0080] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) can be carried out by, but is not limited to, generating a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or improved activity. Specifically, the substitution can be carried out by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used here may further contain a selection marker for confirming whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0081] 6) Introduction of a foreign polynucleotide that exhibits the activity of a polypeptide may be carried out by introducing into a host cell a foreign polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the polypeptide and improving its activity.

[0082] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation within a host cell, or by optimizing the codons of an exogenous polynucleotide so as to achieve optimized transcription and translation within a host cell.

[0083] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed sites may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed sites to be altered or chemically modified.

[0084] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of product produced from the polypeptide.

[0085] For example, the recombinant microorganism capable of producing L-alanine of the present application may be any microorganism that is transformed with a vector and into which a foreign gene encoding the alanine dehydrogenase of the present application, specifically a foreign gene encoding the alanine dehydrogenase derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari, has been introduced, and that produces L-alanine.

[0086] For purposes of this application, the microorganism is defined as, but not limited to, a microorganism that contains an expression vector for expressing the exogenous polynucleotide in a host, thereby enhancing the alanine dehydrogenase protein activity over the endogenous activity.

[0087] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the target polypeptide to be expressed in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0088] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0089] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0090] In the present application, "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that allows it to be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0091] In the present application, "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence directs expression of a coding sequence. Thus, "operably linked" includes a regulatory region of a functional domain having a known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, attached or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target in accordance with the known or desired activity. For example, "operably linked" refers to a polynucleotide sequence operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant polypeptide of the present application.

[0092] "Expression" in this application includes any step involved in producing a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0093] By "expression vector" in this application is meant a linear or circular nucleic acid molecule that contains a coding sequence and regulatory sequences operably linked thereto for the expression thereof.

[0094] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for the expression of a coding sequence. Each regulatory sequence may be a native sequence (having the same origin) relative to the coding sequence, or a foreign sequence (derived from another gene). Examples of such regulatory sequences include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. The smallest unit of such a regulatory sequence includes a promoter and a transcription and translation termination sequence.

[0095] "Recombinant," as used herein with respect to a cell, polynucleotide, polypeptide, or vector, means that the cell, polynucleotide, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or the alteration of a naturally occurring polynucleotide or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express genes that are not found in the native (non-recombinant) form of the cell, or may express naturally occurring genes that are not expressed at all or are aberrantly expressed.

[0096] For example, the L-alanine-producing microorganism may be a microorganism into which a sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or 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 more homology or identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 has been introduced.

[0097] For example, the L-alanine-producing microorganism may be a microorganism that endogenously contains a nucleotide sequence encoding a protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% identity or homology to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0098] For example, the microorganism with improved L-alanine production ability in the present application is a microorganism with improved L-alanine production ability compared to an unmodified microorganism, but is not limited thereto. For example, the unmodified microorganism that is the subject strain for comparison to determine whether the L-alanine production ability is improved is the ATCC13869 strain, but is not limited thereto.

[0099] For example, the microorganism with improved L-alanine producing ability has an L-alanine producing ability that is about 150% or more, specifically about 150% or more, about 200% or more, about 250% or more, about 260% or more, about 270% or more, about 280% or more, about 290% or more, about 300% or more, about 310% or more, about 320% or more, about 325% or more, about 326% or more, about 327% or more, about 328% or more, about 329% or more, about 330% or more, or about The productivity improvement is 331% or more, about 332% or more, about 333% or more, about 334% or more, about 335% or more, or about 336% or more (there is no particular upper limit, and it is, for example, about 1000% or less, about 500% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, or about 340% or less), but any increase in the + value compared to the productivity of the parent strain or unmodified microorganism before mutation is acceptable. In other examples, the recombinant strain with improved L-alanine-producing ability has an L-alanine-producing ability that is about 1.5-fold or more, about 2-fold or more, about 2.5-fold or more, about 2.6-fold or more, about 2.7-fold or more, about 2.8-fold or more, about 2.9-fold or more, about 3-fold or more, about 3.1-fold or more, about 3.2-fold or more, 3.26-fold or more, about 3.3-fold or more, or about 3.36-fold or more (there is no particular upper limit, and for example, about 10-fold or less, about 5-fold or less, about 4-fold or less, about 3.7-fold or less, about 3.5-fold or less, or about 3.4-fold or less) improved compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto.

[0100] For example, the microorganism capable of producing L-alanine may be either a prokaryotic cell or a eukaryotic cell, specifically a prokaryotic cell, such as a microbial strain belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hyphomonas, Chromobacterium, or Norcardia, or a fungi or yeast. Specifically, the microorganisms include strains of the genera Escherichia, Corynebacterium, and Leptospira, and yeasts, and more specifically, strains of the genus Corynebacterium.

[0101] In any of the aforementioned specific examples of microorganisms, the microorganism of the present application may be a Corynebacterium microorganism.

[0102] As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The microorganisms of the present application are, but are not limited to, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium flavescens. Specifically, the microorganisms of the present application are microorganisms of the genus Corynebacterium, more specifically, Corynebacterium glutamicum and Corynebacterium stationis.

[0103] On the other hand, although it is already known that microorganisms of the genus Corynebacterium produce L-alanine, their productivity is very low, and the genes acting on the production mechanism and the principle of the mechanism have not been elucidated. Therefore, the L-alanine-producing microorganisms of the genus Corynebacterium of the present application include all of the following: natural wild-type microorganisms themselves; Corynebacterium microorganisms whose L-alanine-producing ability has been improved by enhancing or reducing the activity of genes involved in the L-alanine production mechanism; and Corynebacterium microorganisms whose L-alanine-producing ability has been improved by introducing or enhancing the activity of an exogenous gene.

[0104] The L-alanine-producing microorganism of the present application is a microorganism in which the introduced alanine dehydrogenase activity has been further enhanced and the L-alanine-producing ability has been improved, but is not limited thereto.

[0105] The microorganism of the present application may be any microorganism in which alanine dehydrogenase activity has been further enhanced by introducing it into the microorganism by various known methods.

[0106] Another aspect of the present application provides a method for producing L-alanine, comprising the step of culturing in a medium an L-alanine-producing Corynebacterium microorganism into which the activity of the alanine dehydrogenase of the present application has been introduced.

[0107] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0108] The term "culturing" as used herein means growing the strain of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the strain selected. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0109] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganisms of the present application, primarily as ingredients, and provides nutrients such as water and growth factors essential for survival and growth. Specifically, the medium and other culture conditions used to culture the strains of the present application may be any medium used for culturing conventional microorganisms, and the microorganisms of the present application can be cultured under aerobic conditions by adjusting the temperature, pH, etc., in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins. For example, a culture medium for Corynebacterium strains is disclosed in Non-Patent Document 16.

[0110] In the present application, examples of carbon sources that can be used 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; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used as long as it is present in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0111] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0112] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0113] Furthermore, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during cultivation of the microorganism of the present application. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0114] In the culture of the present application, the culture temperature is maintained at 27 to 37°C, specifically 30 to 33°C, and the culture is carried out for about 20 to 120 hours, but is not limited thereto.

[0115] In this application, "culture" refers to a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, where the culture solution refers to one containing the specific microorganism, and the culture filtrate refers to one that is substantially free of the specific microorganism (here, this means that the specific microorganism separated by filtration or the like is substantially excluded, but does not mean that the microorganism is completely excluded from the filtrate). The culture may be in any form, such as a liquid, emulsion, or solid. Specifically, for the purposes of this application, the culture may contain L-alanine.

[0116] In this application, "fermentation" refers to the process in which microorganisms use their own enzymes to decompose organic matter, but is not a putrefaction reaction. Fermentation and putrefaction proceed through similar processes, but when useful substances are produced as a result of decomposition, it is called fermentation, and when foul odors or harmful substances are produced, it is called putrefaction.

[0117] In the present application, the method for obtaining a fermented product from the strain is not particularly limited, and the product can be obtained by a method commonly used in the technical field or a similar field.

[0118] In the present application, the term "fermented product" refers not only to the fermented substance itself but also to any kind of substance containing the fermented product generated from the strain, such as a culture medium of the strain in which the strain and culture coexist, a fermented product obtained by filtering the strain from the culture medium, a fermented product obtained by filtering a sterilized strain from the culture medium, an extract obtained by extracting the fermented product or a culture medium containing the fermented product, a diluted solution obtained by diluting the fermented product or an extract thereof, a concentrated solution, a dried product obtained by drying the fermented product or an extract thereof, and a lysate obtained by collecting and disrupting the cells of the strain.

[0119] In the method of the present application, any culture conditions and culture methods known in the art can be used to culture the microorganisms, and those skilled in the art can easily adjust such culture processes depending on the selected strain.

[0120] The L-alanine produced by the culture of the present invention is either secreted into the medium or remains intracellularly.

[0121] In one embodiment, the method for producing L-alanine of the present application may further include a step of providing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), e.g., before the culturing step.

[0122] The method for producing L-alanine of the present application may further include a step of recovering the target substance, specifically L-alanine, from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step may be further included after the culturing step.

[0123] The recovery may involve collecting the target L-alanine using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used. The target substance, specifically L-alanine, can be recovered from the medium or the microorganism using a suitable method known in the art.

[0124] The L-alanine production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-alanine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0125] In the method of the present application, alanine dehydrogenase, the introduction of activity, L-alanine, etc. are as described above.

[0126] Yet another aspect of the present application provides a composition for producing L-alanine, comprising a Corynebacterium microorganism having L-alanine-producing ability, into which activity of the alanine dehydrogenase of the present application has been introduced, a culture of the microorganism, a fermentation product of the microorganism, or a combination of at least two of them.

[0127] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0128] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-alanine, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0129] In one embodiment, each component present in the compositions of the present application is included in a microbiologically effective amount, or in any suitable amount in the production composition.

[0130] In the composition of the present application, the alanine dehydrogenase, the introduction of activity, L-alanine, etc. are as described above.

[0131] Yet another aspect of the present application provides use of a Corynebacterium microorganism having L-alanine production ability, into which the alanine dehydrogenase activity of the present application has been introduced, for L-alanine production.

[0132] In one embodiment, the alanine dehydrogenase may be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari.

[0133] As used herein, alanine dehydrogenase, introduction of activity, L-alanine, etc. are as previously described. [Example]

[0134] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0135] Evaluation of L-alanine production ability of L-alanine-producing microorganisms introduced with exogenous alanine dehydrogenase - 1 Example 1-1: Construction of a vector for introducing a foreign alanine dehydrogenase gene To generate a strain of Corynebacterium glutamicum ATCC13869 carrying exogenous alanine dehydrogenases, vectors were constructed to introduce genes encoding four alanine dehydrogenases.

[0136] Specifically, vectors containing each gene linked to the Pcj7 promoter were constructed.

[0137] First, the intergenic region was selected as the insertion site, and restriction enzymes XbaI and XhoI (tctagactcgag) were inserted into the downstream region of NCgl2195, with 1.0 kb of homologous region on either side. Next, the chromosomal gene of Corynebacterium glutamicum ATCC 13869 was isolated using the Intron G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. Gene fragments (NCgl2195down_A and NCgl2195down_B) were obtained by polymerase chain reaction using the primer pair SEQ ID NO: 22 and SEQ ID NO: 23, and the primer pair SEQ ID NO: 24 and SEQ ID NO: 25, respectively. The polymerase chain reaction conditions were as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. The two fragments were ligated with T4 ligase (New England Biolab, Beverly, AL) to linearized pDCM2 (Patent Document 4) digested with BamHI and SalI. The vector thus constructed was designated pDCM2-ΔNCgl2195down.

[0138] Four chromosomal genes, namely, the ald1 gene (SEQ ID NO: 2) derived from Bacillus licheniformis, the ald2 gene (SEQ ID NO: 4) derived from Bacillus licheniformis, the alaD gene (SEQ ID NO: 6) derived from Bacillus amyloliquefaciens, and the ald2 gene (SEQ ID NO: 8) derived from Laceyella sacchari, were each obtained using Intron's G-spin Total DNA extraction mini kit (Cat. No. 17045).

[0139] Specifically, the DNA was separated according to the protocol provided with the mini kit, and for each gene, a polymerase chain reaction was performed using the primer pair of SEQ ID NOs: 9 and 10, the primer pair of SEQ ID NOs: 11 and 12, the primer pair of SEQ ID NOs: 9 and 13, the primer pair of SEQ ID NOs: 14 and 15, the primer pair of SEQ ID NOs: 9 and 16, the primer pair of SEQ ID NOs: 17 and 18, and the primer pair of SEQ ID NOs: 9 and 19, and the primer pair of SEQ ID NOs: 20 and 21 to obtain four Pcj7 promoter fragments to be ligated to each of the four gene fragments (ald1 (B. li), ald2 (B. li), ald2 (L. sa), and alaD (B. am)). The polymerase chain reaction conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. As a result, polynucleotides of four 364 bp Pcj7 genes and the respective genes ald1(B. li) 1,276 bp, ald2(B. li) 1,283 bp, alaD(B. am) 1,271 bp, and ald2(L. sa) 1,274 bp were obtained.

[0140] The two fragments (Pcj7 and each gene) were ligated with T4 ligase (New England Biolab, Beverly, AL) into linearized pDCM2-ΔNCgl2195down digested with XbaI and XhoI. The resulting vectors were designated pDCM2-ΔNCgl2195down::Pcj7_ald1 (B. li), pDCM2-ΔNCgl2195down::Pcj7_ald2 (B. li), pDCM2-ΔNCgl2195down::Pcj7_alaD (B. am), and pDCM2-ΔNCgl2195down::Pcj7_ald2 (L. sa), respectively.

[0141] [Table 1]

[0142] Example 1-2: Construction of a microorganism into which an exogenous alanine dehydrogenase has been introduced The four vectors prepared in Example 1-1, pDCM2-ΔNCgl2195down::Pcj7_ald1(B.li), pDCM2-ΔNCgl2195down::Pcj7_ald2(B.li), pDCM2-ΔNCgl2195down::Pcj7_alaD(B.am), and pDCM2-ΔNCgl2195down::Pcj7_ald2(L.sa), were each transformed into the parent strain Corynebacterium glutamicum ATCC13869 by electroporation, and then strains in which both the mutant gene and the vector were inserted into the chromosome were selected as the first candidate group from selective medium containing 25 mg / L kanamycin. Subsequently, a second crossover process using homology between the gene on the conventional chromosome and the gene inserted by the vector was performed to obtain the final strain, in which the vector containing the kanamycin resistance gene had been removed from the endogenous ΔNCgl2195down intergenic region on the chromosome. The transformed strains were initially confirmed by PCR using the primer pair SEQ ID NO: 26 and SEQ ID NO: 27, followed by final confirmation by gene sequence analysis. The Pcj7_ald1 (B. li), Pcj7_ald2 (B. li), Pcj7_alaD (B. am), and Pcj7_ald2 (L. sa) transformed strains obtained using the above method were designated CJ0183, CJ0184, CJ0192, and CJ0185.

[0143] [Table 2]

[0144] Example 1-3: Evaluation of L-alanine production ability of L-alanine-producing microorganisms into which exogenous alanine dehydrogenase has been introduced To confirm the L-alanine productivity of Corynebacterium glutamicum CJ0183, CJ0184, CJ0192, and CJ0185 prepared in Example 1-2, they were cultured by the following method.

[0145] The parent strain Corynebacterium glutamicum ATCC13869 and the four mutant strains were inoculated into 25 ml of seed medium in a 250 ml corner baffle flask, and then cultured at 30°C and 200 rpm for 20 hours with shaking to obtain seed cultures. Then, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured at 30°C and 200 rpm for 48 hours to produce L-alanine.

[0146] After the cultivation, the amount of L-alanine produced in the culture medium for each strain tested was measured using high-performance liquid chromatography (Agilent, 1260 Infinity LC system). The compositions of the seed medium and production medium are as follows. The L-alanine concentrations in the culture medium for each strain tested are shown in Table 3. <Seed medium> Glucose 20g / L, Polypeptone 10g / L, Yeast extract 10g / L, (NH4)2SO4 10g / L, Urea 1.5g / L, KH2PO4 5.2g / L, K2HPO4 10.7g / L, d-Biotin 1.8mg / L, Thiamine-HCl 9mg / L, CAPA 9mg / L,NCA 60mg / L,MGSO 40.5g / L <Production medium> CaCO330g / L, Sucrose 57g / L, BM 6g / L, MgSO40.5g / L, (NH4)2SO450g / L, KH2PO41g / L, Yeast extract 2g / L, Ammonium acetate 6.28g / L, d-Biotin 0.05mg / L, Thiamine-HCl 0.1mg / L,MnSO46.7mg / L,FeSO410mg / L

[0147] [Table 3]

[0148] As a result, as shown in Table 3, the parent strain Corynebacterium glutamicum ATCC13869 produced L-alanine at a concentration of 6.1 g / L, while the exogenously introduced strains CJ0183, CJ0184, CJ0192, and CJ0185 produced L-alanine at concentrations of 20.5 g / L, 19.9 g / L, 18.9 g / L, and 15.2 g / L, respectively, confirming that the L-alanine productivity was improved by 336%, 326%, 310%, and 250% compared to the parent strain.

[0149] Therefore, it was confirmed that introduction of the alanine dehydrogenase of the present invention enables high-concentration L-alanine production in Corynebacterium microorganisms. [Example]

[0150] Evaluation of L-alanine production ability of L-alanine-producing microorganisms introduced with exogenous alanine dehydrogenase - 2 Example 2-1: Construction of a recombinant vector containing an additional copy number of a foreign alanine dehydrogenase gene To prepare strains in which one copy of exogenous alanine dehydrogenase was introduced into the CJ0183 and CJ0184 strains prepared in Example 1-2, a copy of the ald1 gene (SEQ ID NO: 2) derived from Bacillus licheniformis, which encodes alanine dehydrogenase, was added in a form linked to the Pcj7 promoter.

[0151] First, the intergenic region was selected as the insertion site, and the NotI and XhoI (gcggccgcctcgag) restriction enzyme sequences were inserted at the NCgl1292 downstream site, with 1.0 kb of homologous region on either side. Next, the chromosomal gene of Corynebacterium glutamicum ATCC 13869 was isolated using the Intron G-spin Total DNA extraction mini kit (Cat. No. 17045) according to the protocol provided with the kit. Gene fragments (NCgl1292down_A and NCgl1292down_B) were obtained by polymerase chain reaction using the primer pair SEQ ID NO:28 and SEQ ID NO:29 and the primer pair SEQ ID NO:30 and SEQ ID NO:31, respectively. The polymerase chain reaction conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. The two fragments were ligated with T4 ligase (New England Biolab, Beverly, AL) to linearized pDCM2 (Patent Document 4) digested with BamHI and SalI. The vector thus constructed was designated pDCM2-ΔNCgl1292down.

[0152] Using the pDCM2-ΔNCgl2195down::Pcj7_ald1(B.li) prepared in Example 1-1 as a template, a polymerase chain reaction was performed using the primer pair of SEQ ID NO: 32 and SEQ ID NO: 33. The conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. As a result, a 1,598-bp Pcj7_ald1(B.li) polynucleotide was obtained. This fragment was ligated with linearized pDCM2-ΔNCgl1292down, which had been digested with NotI and XhoI, using T4 ligase (New England Biolab, Beverly, AL). The vector thus prepared was designated pDCM2-ΔNCgl1292down::Pcj7_ald1(B.li).

[0153] [Table 4]

[0154] Example 2-2: Construction of a microorganism with an increased copy number of an exogenous alanine dehydrogenase gene The parent strains CJ0183 and CJ0184 were transformed with the pDCM2-ΔNCgl1292down::Pcj7_ald1(B. li) vector prepared in Example 2-1 by electroporation. Strains carrying both the mutant gene and the vector integrated into the chromosome were selected as the primary candidate group on selective medium containing 25 mg / L kanamycin. A secondary crossover process using homology between the original chromosomal gene and the vector-inserted gene was then performed to obtain the final strains in which one copy of ald1(B. li) was inserted into the endogenous ΔNCgl1292down gene region on the chromosome and the vector containing the kanamycin resistance gene was deleted. The final strains were initially confirmed by PCR using the primer pair of SEQ ID NO: 34 and SEQ ID NO: 35, followed by final confirmation by gene sequence analysis. The Pcj7_ald1(B. li)-introduced strains obtained using the above method were designated CJ0242 and CJ0229, respectively.

[0155] That is, CJ0242 is a strain in which two copies of B. li ald1 have been added to ATCC13869, and CJ0229 is a strain in which one copy of B. li ald1 and one copy of B. li ald2 have been added to ATCC13869.

[0156] [Table 5]

[0157] Example 2-3: Evaluation of L-alanine production ability of L-alanine-producing microorganisms with added copies of an exogenous alanine dehydrogenase gene To confirm the L-alanine productivity of Corynebacterium glutamicum CJ0242 and CJ0229 prepared in Example 2-2, they were cultured by the following method.

[0158] The parent strain Corynebacterium glutamicum ATCC13869 and five mutant strains were inoculated into 25 ml of seed medium in a 250 ml corner baffle flask, and then cultured at 30°C and 200 rpm for 20 hours with shaking to obtain seed cultures. Then, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured at 30°C and 200 rpm for 48 hours to produce L-alanine.

[0159] After the cultivation was completed, the amount of L-alanine produced in the culture medium of each strain tested was measured using high performance liquid chromatography.

[0160] The compositions of the seed medium and production medium were the same as those in Examples 1 to 3. Table 6 shows the L-alanine concentration in the culture medium for each strain tested.

[0161] [Table 6]

[0162] As a result, as shown in Table 6, the parent strain CJ0183 produced L-alanine at a concentration of 38.2 g / L, while strain CJ0242, in which one copy was additionally introduced into the exogenously introduced strain of the present application, produced L-alanine at a concentration of 40.8 g / L, confirming a 107% increase in L-alanine productivity compared to the parent strain. Also, the parent strain CJ0184 produced L-alanine at a concentration of 37.4 g / L, while strain CJ0229, in which one copy was additionally introduced into the exogenously introduced strain of the present application, produced L-alanine at a concentration of 41.5 g / L, confirming a 111% increase in L-alanine productivity compared to the parent strain.

[0163] Therefore, it was confirmed that introducing an additional copy number into a Corynebacterium microorganism into which the exogenous alanine dehydrogenase of the present invention has been introduced enables the production of L-alanine at a high concentration.

[0164] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. A Corynebacterium microorganism having an ability to produce L-alanine, into which alanine dehydrogenase activity has been introduced, The alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari. Corynebacterium genus microorganisms.

2. The alanine dehydrogenase is encoded by the ald1, ald2, or alaD gene. The Corynebacterium microorganism according to claim 1.

3. The alanine dehydrogenase comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7. The Corynebacterium microorganism according to claim 1.

4. The gene encoding the alanine dehydrogenase comprises the base sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:

8. The Corynebacterium microorganism according to claim 1.

5. The Corynebacterium microorganism is Corynebacterium glutamicum. The Corynebacterium microorganism according to claim 1.

6. The Corynebacterium microorganism has improved L-alanine production ability compared to an unmodified microorganism. The Corynebacterium microorganism according to any one of claims 1 to 5.

7. 1. A method for producing L-alanine, comprising the step of culturing in a medium an L-alanine-producing Corynebacterium microorganism into which alanine dehydrogenase activity has been introduced, The alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari. A method for producing L-alanine.

8. Further comprising a step of recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The method of claim 7.

9. A composition for producing L-alanine, comprising: a Corynebacterium genus microorganism having L-alanine producing ability, into which alanine dehydrogenase activity has been introduced; a culture of said microorganism; a fermentation product of said microorganism; or a combination of at least two of them, The alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari. A composition for producing L-alanine.

10. 1. Use of a Corynebacterium microorganism into which alanine dehydrogenase activity has been introduced for the production of L-alanine, The alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Raceella sacchari. use.

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