Microorganism for producing l-alanine and method for producing l-alanine using same

Abstract

The present disclosure relates to: a Corynebacterium genus microorganism having an L-alanine producing ability into which an alanine dehydrogenase activity has been introduced; a method for producing L-alanine, comprising a step of culturing the microorganism in a culture medium; a composition for producing L-alanine comprising the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of said microorganism for the production of L-alanine.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a Corynebacterium microorganism having L-alanine production ability, in which alanine dehydrogenase activity is introduced; a method of producing L-alanine, which comprises culturing the microorganism in a culture medium; a composition for producing L-alanine, which comprises the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and use of the microorganism for producing L-alanine. BACKGROUND

[0002] L-alanine is an important amino acid, which is widely used in the fields of chemistry, food, medicine, etc. It is an amino acid that produces a sweet taste, and is particularly used as a flavor enhancer and a nutritional supplement in the food industry. As of 2020, the global market value of alanine exceeds $250 million, and the annual production of alanine is about 500 tons (The Expresswire, 2020; Wendisch, 2014).

[0003] In this regard, chemical synthesis or enzymatic conversion is mainly used for the production of L-alanine. Chemical synthesis is achieved by modifying the Strecker synthesis method or the Bucherer-Bergs reaction (Legnani et al., 2021), and enzymatic conversion is achieved by converting L-aspartate to ammonium fumarate (Bucherer-Bergs et al., 1982). Most industrial production is achieved through the enzymatic conversion process. Here, the substrate L-aspartate is produced from petroleum, and the production of fumaric acid also depends on petroleum, which requires high costs, and there is a problem of low yield when petroleum is replaced.

[0004] Accordingly, as an alternative, microbial fermentation can provide an abundant carbon source at low cost. Microorganisms naturally produce L-alanine through transaminase or alanine dehydrogenase. However, there are limitations in using strains of natural origin to produce L-alanine due to low productivity.

[0005] Therefore, there is still a need for research to effectively increase L-alanine production ability. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] The present inventors have confirmed that when alanine dehydrogenase activity is introduced into a microorganism of the genus Corynebacterium, the L-alanine production ability of the microorganism is increased compared to the unmodified microorganism, thereby completing the present disclosure.

[0008] TECHNICAL SOLUTION

[0009] One object of the present disclosure is to provide a microorganism of the genus Corynebacterium having L-alanine production ability, in which alanine dehydrogenase activity is introduced.

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

[0011] In another embodiment, the alanine dehydrogenase can be encoded by an aldl, ald2, or alaD gene.

[0012] In another embodiment, the alanine dehydrogenase can 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 another embodiment, the gene encoding the alanine dehydrogenase can comprise the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0014] As the microorganism according to any one of the above embodiments, the microorganism of the genus Corynebacterium can be Corynebacterium glutamicum.

[0015] As the microorganism according to any one of the above embodiments, the microorganism of the genus Corynebacterium can have increased L-alanine production ability compared to an unmodified microorganism.

[0016] Another object of the present disclosure is to provide a method for producing L-alanine, which comprises culturing a microorganism of the genus Corynebacterium having L-alanine production ability, in which alanine dehydrogenase activity is introduced, in a culture medium.

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

[0018] In another embodiment, the method can further comprise recovering a target substance from the cultured microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or the culture medium.

[0019] Another object of the present disclosure is to provide a composition for producing L-alanine, which comprises: a microorganism of the genus Corynebacterium having L-alanine production ability, in which alanine dehydrogenase activity is introduced, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof.

[0020] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinoplanes thermotolerans.

[0021] Another object of the present disclosure is to provide use of a coryneform microorganism in which alanine dehydrogenase activity is introduced for the production of L-alanine.

[0022] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinoplanes thermotolerans.

[0023] Advantages

[0024] The coryneform microorganism of the present disclosure having L-alanine production ability in which alanine dehydrogenase activity is introduced can produce L-alanine at a high yield, and thus can be effectively used for industrial production of L-alanine. DETAILED DESCRIPTION

[0025] The present disclosure will be described in detail below. Also, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below.

[0026] In addition, those of ordinary skill in the art will recognize or be able to ascertain many equivalents of the specific aspects of the application described herein that are within the scope of the present disclosure. It will be apparent to those of ordinary skill in the art that many alternatives to the embodiments described herein can be employed without departing from the scope of the disclosure.

[0027] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Singular terms shall include pluralities, and plural terms shall include the singular, unless the context clearly dictates otherwise. As used in the specification and the appended claims, the use of "or" can be used to mean "and / or," unless otherwise indicated.

[0028] As used herein, the term "about" can precede a particular value. The term "about" as used herein, includes not only the precise number listed after the term, but also a range around or approximate to the number. Whether a number is around or approximate to a particular number presented can be determined in view of the context in which the number is presented. In one example, the term "about" can refer to a range of -10% to +10% of an index value. In another example, the term "about" can refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0029] As used herein, descriptions such as the terms "first, second, third, …", "i), ii), iii), …" or "(a), (b), (c), (d), …" are used to distinguish similar constituents, and these terms do not mean that the constituents are continuous or sequential. For example, when the terms are used to refer to steps of a method, use, or assay, there can be no time interval between the steps, or they can be performed simultaneously, or they can be performed several seconds, minutes, hours, days, or months apart.

[0030] As used herein, the term "consisting essentially of can mean that unspecified components can be present, provided that the features of the subject matter claimed herein are not materially affected by the presence of the unspecified components.

[0031] As used herein, the term "consisting of refers to a total ratio of 100% of the specified components. The components or features recited after the term "consisting of can be necessary or mandatory. In some embodiments, any other components or non-essential components can be excluded in addition to the components or features recited after the term "consisting of.

[0032] As used herein, the term "comprising / comprising" means the presence of the features, steps, or components recited after the term, and does not exclude the presence or addition of one or more features, steps, or components. The components or features recited herein after the term "comprising / comprising" can be necessary or mandatory. However, in some embodiments, the term can also include any other or non-essential components or features.

[0033] One aspect of the present disclosure provides a microorganism of the genus Corynebacterium having L-alanine production ability, in which alanine dehydrogenase activity is introduced.

[0034] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinopolyspora kivyi.

[0035] The alanine dehydrogenase activity can be defined as increased L-alanine production ability of the microorganism of the present disclosure in which the alanine dehydrogenase activity is introduced, compared to the L-alanine production ability of a natural wild-type microorganism or an unmodified microorganism (e.g., a strain before the introduction of the alanine dehydrogenase activity of the present disclosure), but is not limited thereto.

[0036] For example, the alanine dehydrogenase activity can be evaluated by measuring L-alanine production ability or yield, but is not limited thereto.

[0037] As used herein, the term "alanine dehydrogenase" can refer to an oxidoreductase that catalyzes reversible conversion from pyruvate to L-alanine with coenzyme NAD+ / NADH.

[0038] Specifically, the alanine dehydrogenase of the present disclosure can be a protein having the activity of alanine dehydrogenase encoded by ald1, ald2, or alaD gene, but the type thereof is not particularly limited, as long as the protein has an activity corresponding to the activity of alanine dehydrogenase, and the activity thereof is introduced into a Corynebacterium microorganism, thereby enhancing the L-alanine production ability. The alanine dehydrogenase encoded by ald1, ald2, or alaD gene is known in the art, and the amino acid and polynucleotide sequences of the alanine dehydrogenase can be obtained from GenBank of the known database NCBI, etc., but are not limited thereto.

[0039] In the present disclosure, the alanine dehydrogenase can be one derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinosynnema umbrosa, but is not limited thereto.

[0040] In one example of the present disclosure, the alanine dehydrogenase derived from Bacillus licheniformis can be a protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, but is not limited thereto. The alanine dehydrogenase derived from Bacillus amyloliquefaciens can be a protein comprising the amino acid sequence of SEQ ID NO: 5, but is not limited thereto. The alanine dehydrogenase derived from Actinosynnema umbrosa can be a protein comprising the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0041] For example, the protein having the activity of alanine dehydrogenase in the present disclosure can be a protein comprising the amino acid sequence of the alanine dehydrogenase derived from Bacillus licheniformis (SEQ ID NO: 1 or SEQ ID NO: 3), the amino acid sequence of the alanine dehydrogenase derived from Bacillus amyloliquefaciens (SEQ ID NO: 5), or the amino acid sequence of the alanine dehydrogenase derived from Actinosynnema umbrosa (SEQ ID NO: 7), but is not limited thereto.

[0042] In one example, the alanine dehydrogenase can include 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 thereto, but is not limited thereto, as long as it has alanine dehydrogenase activity. Specifically, the polypeptide having alanine dehydrogenase activity can have, include, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; or have, include, consist of, or consist essentially of 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.

[0043] With regard to the amino acid sequences in the present disclosure, although it is described as a polypeptide "comprising / including" the amino acid sequence described by a specific sequence number, a polypeptide "consisting of" the amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" the amino acid sequence described by a specific sequence number, it is obvious that any protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added, if it has the same or corresponding activity to the protein consisting of the amino acid sequence of the corresponding sequence number, can fall within the scope of the present disclosure. For example, the addition of a sequence upstream or downstream of the amino acid sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof is not excluded, as long as the protein has the same or corresponding activity to the activity of the modified protein, and it is obvious that such sequence addition or mutation falls within the scope of the present disclosure.

[0044] For example, it can be the case of the addition of a sequence, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution that does not change the function of the variant polypeptide of the present disclosure at the N-terminus, the C-terminus, and / or within the amino acid sequence. For example, the polypeptide can be conjugated at the N-terminus with a signal (or leader) sequence involved in protein translocation in a co-translational or post-translational manner. In addition, the polypeptide can also be conjugated with another sequence or linker to identify, purify, or synthesize the polypeptide.

[0045] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitution can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Amino acids can be classified into the following groups:

[0046] In one 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; 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) include arginine, lysine, histidine, glutamic acid, aspartic acid; and amino acids with uncharged side chains (uncharged amino acids; also known as neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan, and tyrosine. In another example, branched-chain amino acids include valine, leucine, and isoleucine. In another example, the 20 amino acids can be classified into 5 groups according to their size, starting from the group of relatively small amino acids, i.e., glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine, but the classification of amino acids is not limited thereto. Generally, conservative substitutions have little or no effect on the activity of a polypeptide.

[0047] Further, the nucleotide sequence encoding alanine dehydrogenase can be a nucleotide sequence encoding a protein exhibiting alanine dehydrogenase activity, and its activity is introduced into a coryneform microorganism, thereby enhancing the L-alanine production ability. For example, it can be a nucleotide sequence encoding 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 Actinobifida kivyi (SEQ ID NO: 7), but is not limited thereto.

[0048] For example, the L-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 can be encoded by a polynucleotide which can have, comprise, consist of, or consist essentially of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or have, comprise, consist of, or consist essentially of a nucleotide sequence having 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% homology or identity to the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, but is not limited thereto. Also, 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 known database such as GenBank of NCBI, but is not limited thereto.

[0049] In the present disclosure, for example, the gene comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 can be used interchangeably with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, the 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 the 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.

[0050] The polynucleotide of the present disclosure can be variously modified in the coding region without changing the amino acid sequence of the alanine dehydrogenase of the present disclosure due to codon degeneracy or in consideration of codons preferred in an organism in which the alanine dehydrogenase of the present disclosure is to be expressed. Therefore, based on codon degeneracy, it is obvious that a polynucleotide which can be translated into a polypeptide consisting of the amino acid sequence of the alanine dehydrogenase of the present disclosure or a polypeptide having homology or identity thereto can also be included. For example, the polynucleotide of the present disclosure can be SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or a degenerate sequence thereof.

[0051] In addition, the polynucleotides of the present disclosure can include probes that can be prepared from known gene sequences, for example, any polynucleotide sequence that hybridizes under stringent conditions to all or a portion of a polynucleotide sequence of the present disclosure to encode an alanine dehydrogenase of the present disclosure, without limitation.

[0052] As used herein, the terms "homology" or "identity" refer to the relatedness of two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms homology and identity can generally be used interchangeably with each other.

[0053] The sequence homology or identity of a conservative polynucleotide or polypeptide can be determined by standard alignment algorithms and can use the default gap penalties established by the program used. Essentially, a homologous or identical sequence is generally expected to hybridize under moderately or highly stringent conditions to all or at least about 50%, 60%, 70%, 80%, or 90% of the length of the sequence. Polynucleotides containing degenerate codons that substitute for the codons in the hybridizing polynucleotide can also be considered.

[0054] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by known computer algorithms, such as the "FASTA" program, using the default parameters in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) using implementation of the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, 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 by using the National Center for Biotechnology Information's BLAST or ClustalW.

[0055] Homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using, for example, the GAP computer program, as disclosed in Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In sum, the GAP program defines homology, similarity or identity as a value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a binary comparison matrix (including a value of 1 for identical and 0 for non-identical) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an extra penalty of 0.10 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 end gaps.

[0056] Further, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing sequence using a Southern hybridization experiment under defined stringent conditions, and suitable hybridization conditions to be defined can be determined by methods within the scope of the present disclosure, which are known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0057] As used herein, "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are disclosed in detail in the literature (e.g., J. Sambrook et al., supra). For example, stringent conditions can include conditions in which polynucleotides having high homology or identity, i.e., polynucleotides having 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 homology or identity, hybridize to each other, while polynucleotides having less than the above homology or identity do not hybridize to each other, or can include washing conditions of ordinary Southern hybridization, i.e., one, in particular two or three, washes at a salt concentration and temperature corresponding to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1xSSC, 0.1% SSD.

[0058] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present disclosure can also include isolated nucleic acid fragments that are complementary to the entire sequence as well as base sequences substantially similar thereto.

[0059] For example, T m Hybridization conditions for a hybridization step having a value of 55°C detect polynucleotides having homology or identity to the polynucleotides of the present disclosure under the conditions described above. In addition, the value of T m The value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to their purposes.

[0060] The appropriate stringency for hybridizing polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0061] As used herein, the term "L-alanine" refers to an L-amino acid having the chemical formula HO2CCH(NH2)CH3, which is an essential amino acid.

[0062] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to insertion of an exogenous gene, or enhancement or inactivation of an endogenous gene activity, and can be a genetically modified microorganism including production of a desired polypeptide, protein, or product. In the present disclosure, "microorganism" and "strain" have the same meaning and can be used interchangeably with each other without limitation.

[0063] For example, the microorganism of the present disclosure can be a microorganism into which alanine dehydrogenase activity is introduced (e.g., a recombinant strain), but is not limited thereto.

[0064] As used herein, the term "microorganism having L-alanine production ability", which is a prokaryotic or eukaryotic microorganism strain capable of producing L-alanine in an organism, can include all of a microorganism in which a parent strain not having L-alanine production ability has been given L-alanine production ability, or a microorganism having endogenous L-alanine production ability. L-alanine production ability can be given or enhanced by improving a species.

[0065] As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, and can refer to a wild-type strain or a natural-type strain itself, or a strain before its properties are changed due to genetic modification by natural or artificial factors. The "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism", "parent strain before mutation", "wild-type microorganism", "reference microorganism", or "standard microorganism". In the present disclosure, the unmodified microorganism can refer to a strain into which alanine dehydrogenase activity is not introduced, or a strain before introduction of alanine dehydrogenase, but is not limited thereto. In addition, in the present disclosure, the unmodified microorganism can be a microorganism not including 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.

[0066] For the purpose of the present disclosure, the microorganism of the present disclosure can include all microorganisms capable of producing the desired L-alanine by introducing the activity of L-alanine dehydrogenase. For example, the microorganism of the present disclosure is characterized in that the activity of alanine dehydrogenase is introduced, thereby increasing the L-alanine production ability, and can be a genetically modified microorganism or a recombinant microorganism, but is not limited thereto. Specifically, the recombinant strain having increased L-alanine production ability can be a microorganism having increased L-alanine production ability compared to a natural wild-type microorganism or an unmodified microorganism having endogenous activity of alanine dehydrogenase, but is not limited thereto.

[0067] In one example, the microorganism having L-alanine production ability, which is a prokaryotic or eukaryotic microorganism strain capable of producing L-alanine in an organism, can include all of the following: a microorganism having endogenous L-alanine production ability, or a microorganism in which a parent strain not having L-alanine production ability has been given L-alanine production ability by the activity of the alanine dehydrogenase introduced in the present disclosure. The L-alanine production ability can be given or enhanced by improving the species.

[0068] The microorganism of the present disclosure can include all microorganisms into which the activity of alanine dehydrogenase is introduced by various known methods.

[0069] As used herein, the term "introducing" activity means that a gene that a microorganism does not originally have is expressed in the microorganism, so that the microorganism exhibits the activity of a specific protein, or the activity of a polypeptide is enhanced, increased, or improved compared to the endogenous activity or the activity before modification of the corresponding protein. For example, the term can mean that a polynucleotide encoding a specific protein is introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, thereby exhibiting the activity of a specific protein.

[0070] "Endogenous activity" means the activity of a specific polypeptide that a parent strain before transformation or an unmodified microorganism originally has when the trait is changed due to genetic variation caused by natural or artificial factors. The endogenous activity can also be used interchangeably with "activity before modification."

[0071] As used herein, the term "enhancement" of the activity of a polypeptide means that the activity of a polypeptide is increased compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, up-regulation, overexpression, increase, etc.

[0072] In particular, activation, enhancement, up-regulation, overexpression, increase can include exhibiting an activity that was not originally present, or an activity that is enhanced compared to the endogenous activity or the activity prior to the modification. A polypeptide activity is "enhanced", "up-regulated", "overexpressed", or "increased" compared to the endogenous activity means that the polypeptide activity is enhanced compared to the activity and / or concentration (expression level) of the particular polypeptide that the parent strain prior to transformation or the unmodified microorganism originally had. For example, the term "exhibiting an activity that was not originally present" can refer to "introducing a protein", but is not limited thereto.

[0073] An enhancement of a polypeptide activity compared to the endogenous activity means that the polypeptide activity is enhanced compared to the activity and / or concentration (expression level) of the particular polypeptide that the parent strain prior to transformation or the unmodified microorganism originally had.

[0074] In one example, enhancement can mean exhibiting an activity of the respective protein that was not originally present, or its activity or concentration is generally enhanced by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000%, or about 2000% or more, but is not limited thereto.

[0075] An enhancement of a polypeptide activity can be achieved by introducing an exogenous polypeptide or enhancing the activity of an endogenous polypeptide. Whether a polypeptide activity is enhanced can be evidenced by an increase in the activity level of the respective polypeptide, its expression level, or the amount of product produced by the respective polypeptide. As used herein, the term "about" refers to a range that includes all ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values equivalent to the value immediately following the term "about" or within a similar range, but is not limited thereto.

[0076] An enhancement can be achieved by introducing an exogenous polypeptide or enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether a polypeptide activity is enhanced can be evidenced by an increase in the activity level of the respective polypeptide, its expression level, or the amount of product produced by the respective polypeptide.

[0077] An enhancement of a polypeptide activity can be achieved by various methods well known in the art and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism prior to the modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods of molecular biology, but the methods 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.).

[0078] Specifically, the enhancement of the polypeptide of the present disclosure can be achieved by:

[0079] 1) increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0080] 2) modifying the expression regulatory region of the gene encoding the polypeptide on the chromosome (e.g., inducing a modification in the expression regulatory region, replacing with a sequence having stronger activity, or inserting a sequence having stronger activity);

[0081] 3) modifying the nucleotide sequence of the coding initiation codon or the 5'-UTR of the transcript of the gene encoding the polypeptide;

[0082] 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced;

[0083] 5) modifying the polynucleotide sequence encoding the polypeptide so that the polypeptide activity is enhanced (e.g., modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the polypeptide activity);

[0084] 6) introducing an exogenous polypeptide or an exogenous polynucleotide encoding the polypeptide that exhibits the activity of the polypeptide;

[0085] 7) codon-optimizing the polynucleotide encoding the polypeptide;

[0086] 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying the exposed sites therefrom; or

[0087] 9) a combination of two or more selected from the above 1) to 8), but not particularly limited thereto.

[0088] The method of the above 1) increasing the copy number of the polynucleotide encoding the polypeptide in the cell can be achieved by introducing a vector into the host cell, which is operably linked to the polynucleotide encoding the polypeptide and is capable of replication and functioning independently of the host cell. Alternatively, the method can be achieved by introducing one or two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. The introduction into the chromosome can be performed by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited thereto. The vector is as described above.

[0089] The method of 2) replacing the expression regulatory region (or expression regulatory sequence) of the gene encoding the polypeptide on the chromosome with a sequence having strong activity can be further enhanced by, for example, introducing a modification in the sequence by deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof to further enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence having stronger activity. The expression regulatory region can include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, etc. In one example, the method can include replacing the original promoter with a strong promoter, but is not limited thereto.

[0090] Examples of known strong promoters can include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda 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 the strong promoter is not limited thereto.

[0091] The method of 3) modifying the nucleotide sequence encoding the start codon of the transcript of the gene encoding the polypeptide or the 5'-UTR can be achieved by, for example, replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0092] The methods of 4) and 5) modifying the amino acid sequence or the polynucleotide sequence can be achieved by inducing a modification in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence having stronger activity, or an amino acid sequence or polynucleotide sequence modified to enhance the activity, but is not limited thereto. Specifically, the replacement can be performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein can further include a selection marker to confirm insertion into the chromosome. The selection marker is as described above.

[0093] The method of 6) introducing an exogenous polynucleotide exhibiting the activity of the polypeptide can be achieved by introducing an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The exogenous polynucleotide can be used without limitation regardless of its origin or sequence, as long as it exhibits the same / similar activity as the polypeptide. The introduction can be performed by appropriately selecting a transformation method known in the art by those of ordinary skill in the art, and the expression of the introduced polynucleotide in the host cell can produce the polypeptide, thereby increasing its activity.

[0094] The method of 7) codon optimization of the polynucleotide encoding the polypeptide can be achieved by codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or by optimization of the codon so that optimized transcription and translation of the exogenous polynucleotide can be achieved in the host cell.

[0095] The method of 8) analyzing the tertiary structure of the polypeptide and thereby selecting and modifying or chemically modifying the exposed site can be achieved by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information to determine template protein candidates according to the degree of sequence similarity, and thus confirming the structure based on the information to select and transform or modify the exposed site to be modified or chemically modified.

[0096] Such enhancement of the activity of the polypeptide can mean an increase in the activity or concentration (expression level) of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type strain or the microorganism before modification, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0097] In one example, the recombinant microorganism having L-alanine production ability can include all microorganisms that can be transformed by a vector and thus can produce L-alanine by introducing an exogenous gene encoding the alanine dehydrogenase of the present disclosure, particularly an exogenous gene encoding the alanine dehydrogenase derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinopolyspora thalpophilus.

[0098] For the purpose of the present disclosure, the microorganism can be a microorganism in which the activity of alanine is enhanced compared to the endogenous activity by including an expression vector for expressing an exogenous polynucleotide in a host cell, but is not limited thereto.

[0099] The vector of the present disclosure can include a DNA construct containing a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression regulatory region (expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host cell. The expression regulatory region can include a promoter capable of initiating transcription, any operator sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription and translation termination. Once transformed into a suitable host cell, the vector can replicate independently of the host genome or function, or can be integrated into the genome thereof.

[0100] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of the commonly used vector can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as bacteriophage vectors or cosmid vectors; those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0101] In one example, the polynucleotide encoding the target polypeptide can be inserted into a chromosome through a vector for intracellular chromosomal insertion. Inserting the polynucleotide into the chromosome can be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector can further include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm whether the target nucleic acid molecule is inserted, and a marker that provides a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression) can be used. Only cells expressing the selection marker can survive in an environment treated with a selection agent or show a different phenotype, and thus transformed cells can be selected.

[0102] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a polypeptide of interest into a host cell or microorganism, such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be integrated into the chromosome of the host cell and located therein or can be located extrachromosomally, as long as the transformed polynucleotide can be expressed in the host cell, both of which are included. In addition, the polynucleotide can include DNA and / or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression thereof. The expression cassette can generally contain a promoter operably linked to the polynucleotide, a transcription terminator, a ribosome binding site, or a translation terminator. The expression cassette can be in the form of a self-replicating expression vector. In addition, the polynucleotide can be introduced into the host cell as is and can be operably linked to sequences required for expression in the host cell, but is not limited thereto.

[0103] As used herein, the term "operably linked" refers to the configuration in which a regulatory sequence is placed in the appropriate position relative to the coding sequence of the gene so as to control the expression of the coding sequence. Thus, the term "operably linked" includes attachment or linkage between a regulatory region (such as a promoter, stop codon, signal sequence, or enhancer) having a known or desired activity and a target (gene or polypeptide) such that the expression, secretion, or function of the target can be modulated according to the known or desired activity. For example, it can refer to the functional linkage of a polynucleotide sequence with a promoter sequence that initiates and mediates transcription of the polynucleotide encoding a target variant polypeptide of the present disclosure.

[0104] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0105] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule, including a coding sequence and an operably linked regulatory sequence for expression thereof.

[0106] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence can be native (derived from the same source) or foreign (derived from a different gene) to the coding sequence. Examples of the regulatory sequence can 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 transcription and translation termination. The minimum unit of the regulatory sequence can include a promoter and a sequence for termination of transcription and translation.

[0107] As used herein, the term "recombinant" 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 alteration of a native polynucleotide or polypeptide, or that the cell is derived from such a modified cell. Thus, for example, a recombinant cell can express genes that are not found in cells of the same species under natural (non-recombinant) conditions, or can express native genes that are otherwise expressed at unusually high levels, at levels inappropriate to their normal physiological milieu, or at levels that vary with respect to a natural physiological rhythm.

[0108] For example, the microorganism for producing L-alanine can 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 a protein consisting of 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 SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 has been introduced.

[0109] For example, the microorganism for producing L-alanine can be a microorganism endogenously comprising a nucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, and a nucleotide sequence having 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% 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.

[0110] In one example, the microorganism of the present disclosure having increased L-alanine production capacity can be a microorganism having increased L-alanine production capacity compared to an unmodified microorganism, but is not limited thereto. In one example, the unmodified microorganism, which is a target strain for comparison of the increase in L-alanine production capacity, can be the ATCC13869 strain, but is not limited thereto.

[0111] In one example, the microorganism having increased L-alanine productivity can have L-alanine productivity that is increased by 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, about 331% or more, about 332% or more, about 333% or more, about 334% or more, about 335% or more, or about 336% or more (the upper limit is not particularly limited, 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 not limited thereto, as long as the + value is increased compared to the productivity of the parent strain before modification or the unmodified microorganism. In another example, the microorganism having increased L-alanine productivity can have L-alanine productivity that is increased by 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 (the upper limit is not particularly limited, 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), but not limited thereto.

[0112] In one example, the microorganism having L-alanine-producing ability can be a prokaryotic cell or a eukaryotic cell, but can be particularly a prokaryotic cell. The prokaryotic cell can include, for example, a microbial strain belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomononas, Chromobacterium, and Norcardia, or a fungus or a yeast. Specifically, the microorganism can be a microbial strain belonging to the genera Escherichia, Corynebacterium, Leptospira, and a yeast. More specifically, it can be a microbial strain belonging to the genus Corynebacterium.

[0113] As the microorganism according to any one of the above-described embodiments, the microorganism of the present disclosure can be a Corynebacterium microorganism.

[0114] In one example, the microorganism of the present disclosure can 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 microorganism of the present disclosure can be a Corynebacterium microorganism, more specifically, Corynebacterium glutamicum or Corynebacterium stationis, but is not limited thereto.

[0115] Meanwhile, although it is known that Corynebacterium microorganisms can produce L-alanine, they have very low L-alanine production ability, and the genes acting on the production mechanism or the principle of the mechanism have not yet been known. Therefore, the Corynebacterium microorganism having L-alanine production ability of the present disclosure can include all of: a wild-type Corynebacterium microorganism itself; a Corynebacterium microorganism in which the activity of a gene related to the L-alanine production mechanism is enhanced or inactivated to have L-alanine production ability; or a Corynebacterium microorganism in which the activity of an exogenous gene is introduced or enhanced to have L-alanine production ability.

[0116] The microorganism having L-alanine production ability of the present disclosure can be a microorganism having enhanced L-alanine production ability by further enhancing the activity of the introduced alanine dehydrogenase, but is not limited thereto.

[0117] The microorganism of the present disclosure can include all of the microorganisms in which the activity of the introduced alanine dehydrogenase is further enhanced by various methods in the art.

[0118] Another aspect of the present disclosure provides a method for producing L-alanine, which comprises culturing a coryneform microorganism of the present disclosure having an L-alanine production ability into which alanine dehydrogenase activity is introduced in a culture medium.

[0119] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinoplanes thermotolerans.

[0120] As used herein, the term "culturing" refers to growing the strains of the present disclosure under suitably controlled environmental conditions. The culturing process of the present disclosure can be performed under suitable culture media and culture conditions known in the art. Such a culturing process can be easily adjusted for use by those skilled in the art depending on the strain to be selected. Specifically, the culturing can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto.

[0121] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for culturing the microorganisms of the present disclosure as a main component, which provides nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions for culturing the strains of the present disclosure can be any culture medium used for conventional culturing of microorganisms without any particular limitation. However, the microorganisms of the present disclosure can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin under aerobic conditions, while adjusting the temperature, pH, etc. For example, the culture medium for coryneform microorganisms can be found in the literature [“Manual of Methods for General Bacteriology” (Washington D.C., USA, 1981) of the American Society for Bacteriology].

[0122] In the present disclosure, the carbon source can include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source can include natural organic nutrients such as starch hydrolysate, molasses, jaggery paste, rice bran, cassava, sugar cane molasses, and corn syrup, etc. Specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugar) can be used, and in addition, various other carbon sources in a suitable amount can be used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0123] The nitrogen source can include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0124] The phosphorus source can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt, etc. Examples of inorganic compounds can include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors, etc. can be included. These components or precursors can be added to the culture medium in a batch or continuous manner, but the phosphorus source is not limited thereto.

[0125] In addition, in the process of culturing the microorganism of the present disclosure in an appropriate manner, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, etc. In addition, an antifoaming agent such as a fatty acid polyethylene glycol ester can be used to prevent the formation of bubbles during the culturing process. In addition, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen, or carbon dioxide or no gas can be injected to maintain anaerobic or microaerophilic conditions, but the gas is not limited thereto.

[0126] The temperature during the culturing of the present disclosure can be in the range of 27°C to 37°C, specifically 30°C to 33°C, and the culturing can continue for about 20 hours to 120 hours, but is not limited thereto.

[0127] As used herein, the term "culture product" refers to a culture broth, a concentrated culture broth, a dried product of the culture broth, a culture filtrate, a concentrated culture filtrate, or a dried product of the culture filtrate obtained by culturing a specific microorganism in a culture medium, and refers to the culture broth can include the specific microorganism, while the culture filtrate substantially does not include the specific microorganism (specifically, it substantially refers to excluding the specific microorganism isolated by filtration, etc., but does not mean that the microorganism is completely excluded from the filtrate). The dosage form of the culture product is not limited, and can be, for example, a liquid, an emulsion, or a solid. Specifically, for the purpose of the present disclosure, the culture product can include L-alanine.

[0128] As used herein, the term "fermentation" refers to the process in which a microorganism does not spoil in the decomposition of organic matter using its own enzymes. Fermentation and spoilage reactions are carried out by similar processes, but when decomposition produces useful substances, it is called fermentation, and when it produces odor or harmful substances, it is called spoilage.

[0129] In the present disclosure, the method of obtaining a fermentation product from a strain is not particularly limited, and can be obtained according to a method commonly used in the art or a similar field.

[0130] As used herein, the term "fermentation product" can include not only the fermentation material itself, but also all kinds of materials, including a fermentation product produced by a strain, such as a strain culture medium coexisting with a strain and a culture; a fermentation product produced from a culture medium; a fermentation product obtained by filtering a strain from a culture medium; a fermentation product obtained by sterilizing a strain from a culture medium and filtering the strain, an extract obtained by extracting a fermentation product or a culture medium containing a fermentation product; a dilution solution obtained by diluting a fermentation product or an extract thereof; a concentrated solution; a dried product obtained by drying a fermentation product or an extract thereof; and a lysate obtained by collecting and lysing a strain cell, and the like.

[0131] In the method of the present disclosure, the culture of a microorganism can be performed by any culture condition and culture method known in the art. Such a culture process can be easily adjusted and used by a person skilled in the art according to the selected strain.

[0132] L-alanine produced by the culture of the present disclosure can be released into the culture medium or retained in the cell.

[0133] In one embodiment, the method of producing L-alanine of the present disclosure can further include, for example, a step of preparing a microorganism of the present disclosure before the culturing step, a step of preparing a culture medium for culturing a strain, or a combination thereof (in any order).

[0134] The method of producing L-alanine of the present disclosure can further include a step of recovering a desired substance, particularly L-alanine, from the cultured microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a culture medium. The recovery step can also include after the culturing step.

[0135] In the recovery step, the desired L-alanine can be collected using a method of culturing a microorganism of the present disclosure, for example, according to a batch culture, a continuous culture, or a fed-batch culture method, using a suitable method known in the art. For example, a method such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and the like, or a combination thereof, and a suitable method known in the art can be used to recover a desired substance, particularly L-alanine, from a culture medium or a microorganism.

[0136] Further, the method for producing L-alanine of the present disclosure can further include a purification step, which can be performed using a suitable method known in the art. In one example, when the method for producing L-alanine of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or intermittently without regard to the order, or can be performed simultaneously, or can be integrated into one step, but the method is not limited thereto.

[0137] In the method of the present disclosure, the alanine dehydrogenase, the introduced activity, and the L-alanine, etc. are as described in other aspects above.

[0138] Another aspect of the present disclosure provides a composition for producing L-alanine, comprising: a coryneform microorganism having L-alanine production ability of the present disclosure in which alanine dehydrogenase activity is introduced, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof.

[0139] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinoplanes thermotolerans.

[0140] The composition of the present disclosure can further include any suitable excipient generally used in a composition for producing L-alanine, and such an excipient can include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizer, or an isotonic agent, etc., but is not limited thereto.

[0141] In one embodiment, each component present in the composition of the present disclosure can be included in a microbiologically effective amount, or in an amount that can be appropriately present in a composition for production.

[0142] In the composition of the present disclosure, the alanine dehydrogenase, the introduced activity, and the L-alanine, etc. are as described in other aspects above.

[0143] Another aspect of the present disclosure provides use of a coryneform microorganism having L-alanine production ability of the present disclosure in which alanine dehydrogenase activity is introduced for producing L-alanine.

[0144] In one embodiment, the alanine dehydrogenase can be derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Actinoplanes thermotolerans.

[0145] In the use of the present disclosure, the alanine dehydrogenase, the introduced activity, and the L-alanine, etc. are as described in other aspects above.

[0146] Mode for carrying out the invention

[0147] The present disclosure will be described in detail by way of examples below. However, the examples are given for illustrative purposes only and, as such, the scope of the present disclosure is not intended to be limited to these examples or by these examples. Meanwhile, those skilled in the technical field of the present disclosure or a similar technical field can fully understand and easily implement technical features not described herein.

[0148] Example 1: Evaluation of L-alanine production ability of L-alanine production microorganism into which exogenous alanine dehydrogenase is introduced - 1

[0149] Example 1-1: Construction of vector for introduction of exogenous alanine dehydrogenase gene

[0150] In order to construct a strain into which exogenous alanine dehydrogenase is introduced into Corynebacterium glutamicum ATCC13869, a vector was constructed to introduce genes encoding four types of alanine dehydrogenase.

[0151] Specifically, a vector containing each gene linked to the Pcj7 promoter was constructed.

[0152] First, an intergenic region was selected as a copy insertion site, and the sequences of restriction enzymes XbaI and XhoI (tctagactcgag) were inserted in the downstream region of NCgl2195, and a homologous region of 1.0 kb was inserted on each side. Thereafter, the chromosomal gene of the Corynebacterium glutamicum ATCC13869 strain was isolated using the G-spin total DNA extraction mini kit (Cat. No. 17045, Promega) according to the protocol provided in the kit, and PCR was performed using the primer pairs of SEQ ID NO: 22 and 23 and the primer pairs of SEQ ID NO: 24 and 25, to obtain gene fragments (NCgl2195 down_A, NCgl2195 down_B), respectively. PCR was performed under the following conditions: denaturation at 95℃ for 5 minutes, followed by 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and polymerization at 72℃ for 30 seconds, and then polymerization at 72℃ for 5 minutes. The two gene fragments cut with BamHI and SalI restriction enzymes were ligated to linear pDCM2 (Korean Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-ΔNCgl2195 down.

[0153] Four types of chromosomal genes were obtained using the G-spin Total DNA Extraction Mini Kit (Cat. No. 17045, Introns): the ald1 gene (SEQ ID NO: 2) from Bacillus licheniformis, the ald2 gene (SEQ ID NO: 4) from Bacillus licheniformis, the alaD gene (SEQ ID NO: 6) from Bacillus amyloliquefaciens, or the ald2 gene (SEQ ID NO: 8) from Lespedeza thermophila.

[0154] Specifically, genes were isolated according to the protocol provided in the mini kit. Each gene was subjected to PCR using primer pairs SEQ ID NO: 9 and 10 and SEQ ID NO: 11 and 12, primer pairs SEQ ID NO: 9 and 13 and SEQ ID NO: 14 and 15, primer pairs SEQ ID NO: 9 and 16 and SEQ ID NO: 17 and 18, and primer pairs SEQ ID NO: 9 and 19 and SEQ ID NO: 20 and 21, respectively, to obtain four Pcj7 promoter fragments that could be ligated to each of the four gene fragments, and the four gene fragments (ald1(B. li), ald2(B. li), ald2(L. sa), alaD(B. am)) ligated to each of the four gene fragments. PCR was performed under the following conditions: 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, four 364 bp Pcj7 fragments and corresponding gene polynucleotides were obtained (1,276 bp ald1 (B. li), 1,283 bp ald2 (B. li), 1,271 bp aldD (B. am) and 1,274 bp ald2 (L. sa)).

[0155] Two gene fragments (Pcj7 and each gene) digested with XbaI and XhoI restriction enzymes were ligated into linear pDCM2-ΔNCgl2195 vectors using T4 ligase (New England Biolabs, Beverly, MA). The resulting vectors were named pDCM2-ΔNCgl2195::Pcj7_ald1 (B. li), pDCM2-ΔNCgl2195::Pcj7_ald2 (B. li), pDCM2-ΔNCgl2195::Pcj7_alaD (B. am), and pDCM2-ΔNCgl2195::Pcj7_ald2 (L. sa).

[0156] [Table 1]

[0157]

[0158] Example 1-2: Construction of microorganism into which exogenous alanine dehydrogenase is introduced

[0159] Each of the four types of vectors constructed 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), was transformed into the parent strain Corynebacterium glutamicum ATCC 13869 by electroporation, and then strains in which the variant gene and the vector were inserted into the chromosome were selected in a selection medium containing 25 mg / L kanamycin as primary candidates. Thereafter, using homology between the gene present on the chromosome and the gene inserted by the vector, the strains inserted into the endogenous ΔNCgl2195down intergenic region on the chromosome by a secondary crossover process. The final strains in which the vector containing the kanamycin resistance gene was removed were obtained. The resulting strains were preliminarily detected by PCR using the primer pair of SEQ ID NO: 26 and SEQ ID NO: 27, and finally confirmed by sequencing. The strains into which Pcj7_ald1(B.li), Pcj7_ald2(B.li), Pcj7_alaD(B.am), and Pcj7_ald2(L.sa) were thus obtained were designated as CJ0183, CJ0184, CJ0192, and CJ0185, respectively.

[0160] [Table 2]

[0161]

[0162] Example 1-3: Evaluation of L-alanine production ability of L-alanine production microorganism into which exogenous alanine dehydrogenase is introduced

[0163] To confirm the L-alanine production ability of Corynebacterium glutamicum CJ0183, CJ0184, CJ0192, and CJ0185 constructed in Example 1-2 described above, the strains were cultured as follows:

[0164] Corynebacterium glutamicum ATCC 13869 and four types of variant strains were inoculated into 250 mL corner-baffled flasks containing 25 mL of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm to obtain a seed culture solution. Thereafter, 1 mL of the seed culture solution was inoculated into 250 mL corner-baffled flasks containing 24 mL of production medium and cultured at 30°C for 48 hours with shaking at 200 rpm to prepare L-alanine.

[0165] After completion of the culture, the yield of L-alanine was measured using high-performance liquid chromatography (Agilent, 1260 Infinity LC system), and the yield of L-alanine in the culture solution of each test strain was measured. The compositions of the seed medium and the production medium were as follows, and the L-alanine concentration in the culture solution of each test strain was as shown in Table 3 below.

[0166] <Seed medium>

[0167] 20 g / L glucose (anhydrous glucose), 10 g / L polyproline peptone, 10 g / L yeast extract, 10 g / L (NH4)2SO4, 1.5 g / L urea, 5.2 g / L KH2PO4, 10.7 g / L K2HPO4, 1.8 mg / L d-biotin, 9 mg / L thiamine hydrochloride, 9 mg / L CAPA, 60 mg / L NCA, 0.5 g / L MGSO4

[0168] <Production medium>

[0169] 30 g / L CaCO3, 57 g / L sucrose, 6 g / L BM, 0.5 g / L MgSO4, 50 g / L (NH4)2SO4, 1 g / L KH2PO4, 2 g / L yeast extract, 6.28 g / L ammonium acetate, 0.05 mg / L d-biotin, 0.1 mg / L thiamine hydrochloride, 6.7 mg / L MnSO4, 10 mg / L FeSO4

[0170] [Table 3]

[0171]

[0172] As shown in Table 3, the concentration of L-alanine produced by the parental strain Corynebacterium glutamicum ATCC13869 was 6.1 g / L. However, the concentrations of L-alanine produced by the strains CJ0183, CJ0184, CJ0192 and CJ0185, which were introduced with exogenous alanine dehydrogenase as described in this disclosure, were 20.5 g / L, 19.9 g / L, 18.9 g / L and 15.2 g / L, respectively. This confirms that the L-alanine production capacity increased by 336%, 326%, 310% and 250% compared with the parental strain.

[0173] Therefore, it has been confirmed that L-alanine can be produced at high concentrations in Corynebacterium microorganisms by introducing alanine dehydrogenase, as disclosed in this invention.

[0174] Example 2: Evaluation of L-alanine production capacity of L-alanine-producing microorganisms introduced with exogenous alanine dehydrogenase -2

[0175] Example 2-1: Constructing a recombinant vector containing an additional copy of the exogenous alanine dehydrogenase gene

[0176] To construct strains in which an additional copy of the exogenous alanine dehydrogenase was introduced into the CJ0183 and CJ0184 strains constructed in Examples 1-2, an additional copy of the gene encoding the alanine dehydrogenase derived from the ald1 gene (SEQ ID NO: 2) of Bacillus licheniformis was introduced by linking it to the Pcj7 promoter.

[0177] First, the intergenic region was selected as the copy insertion site. Restriction enzyme sequences NotI and XhoI (gcggccgcctcgag) were inserted downstream of NCgl1292, with 1.0 kb homologous regions inserted on each side. Subsequently, following the protocol provided in the kit, the chromosomal genes of *Corynebacterium glutamicum* strain ATCC13869 were isolated using the G-spin Total DNA Extraction Mini Kit (Cat. No. 17045, Introns). PCR was performed using primer pairs SEQ ID NO: 28 and 29 and primer pairs SEQ ID NO: 30 and 31 to obtain gene fragments (NCgl1292_A and NCgl1292_B), respectively. PCR was performed under the following conditions: 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. Two gene fragments digested with BamHI and SalI restriction enzymes were ligated into a linear pDCM2 vector (Korean Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-ΔNCgl1292.

[0178] PCR was performed using the primer pair of SEQ ID NO: 32 and 33 based on pDCM2-ΔNCgl2195 down::Pcj7_ald1 (B. li) constructed in Example 1-1 above as a template. PCR was performed under the following conditions: 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 Pcj7_ald1 (B. li) polynucleotide of 1,598 bp was obtained. The fragment cut with Notl and Xhol restriction enzymes was ligated to linear pDCM2-ΔNCgl1292 down using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-ΔNCgl1292 down::Pcj7_ald1 (B. li).

[0179] [Table 4]

[0180]

[0181] Example 2-2: Construction of microorganisms having additional copies of exogenous alanine dehydrogenase genes

[0182] The pDCM2-ΔNCgl1292 down::Pcj7_ald1 (B. li) vector constructed in Example 2-1 was transformed into each of the parent strains CJ0183 and CJ0184 by electroporation, and then strains in which the variant gene and the vector insertion into the chromosome were selected in a selection medium containing 25 mg / L kanamycin as primary candidates. Thereafter, using homology between the gene present on the chromosome and the gene inserted by the vector, one copy of ald1 (B. li) was additionally introduced into the endogenous ΔNCgl1292 down intergenic region on the chromosome through a second exchange process. The final strain in which the vector containing the kanamycin resistance gene was removed was obtained. The final strain was primarily detected by PCR using the primer pair of SEQ ID NO: 34 and SEQ ID NO: 35, and finally confirmed by sequencing. The strains in which an additional copy of Pcj7_ald1 (B. li) was introduced thus obtained were named CJ0242 and CJ0229, respectively. That is, CJ0242 is a strain to which two copies of B. li ald1 were added with respect to ATCC13869, and CJ0229 is a strain to which one copy of B. li ald1 and one copy of B. li ald2 were added with respect to ATCC13869.

[0183] [Table 5]

[0184]

[0185] Example 2-3: Evaluation of L-alanine production ability of L-alanine production microorganism having additional copy of exogenous alanine dehydrogenase gene

[0186] To confirm the L-alanine production ability of Corynebacterium glutamicum CJ0242 and CJ0229 constructed in Example 2-2 above, the strains were cultured as follows:

[0187] Corynebacterium glutamicum ATCC13869 and five types of variant strains were inoculated into 250 mL of a baffled flask containing 25 mL of seed culture medium and cultured at 30°C for 20 hours with shaking at 200 rpm to obtain a seed culture solution. Thereafter, 1 mL of the seed culture solution was inoculated into a 250 mL baffled flask containing 24 mL of production culture medium and cultured at 30°C for 48 hours with shaking at 200 rpm to prepare alanine.

[0188] After completion of the culture, the yield of L-alanine was measured using high performance liquid chromatography, and the yield of L-alanine in the culture solution of each test strain was measured. The composition of the seed culture medium and the production culture medium was as follows, and the L-alanine concentration in the culture solution of each test strain is shown in Table 6 below.

[0189] [Table 6]

[0190]

[0191] As a result, as shown in Table 6 above, the parent strain CJ0183 produced L-alanine at a concentration of 38.2 g / L, but the CJ0242 strain, in which one additional copy was introduced into a strain into which an exogenous alanine dehydrogenase was introduced according to the present disclosure, produced L-alanine at a concentration of 40.8 g / L, confirming that the L-alanine production ability was increased by 107% compared to the parent strain. In addition, the parent strain CJ0184 produced L-alanine at a concentration of 37.4 g / L, but the CJ0229 strain, in which one additional copy was introduced into a strain into which an exogenous alanine dehydrogenase was introduced according to the present disclosure, produced L-alanine at a concentration of 41.5 g / L, confirming that the L-alanine production ability was increased by 111% compared to the parent strain.

[0192] Accordingly, it was confirmed that L-alanine can be produced at a high concentration by introducing an additional copy into the Corynebacterium microorganism of the present disclosure into which an exogenous alanine dehydrogenase was introduced.

[0193] According to the foregoing, it will be understood by those skilled in the art that the present disclosure can be implemented in other specific forms without modifying the technical concept or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are merely for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Rather, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalent replacements, and other embodiments that can be included in the spirit and scope of the present disclosure defined by the appended claims.

Claims

1. A microorganism of the genus Corynebacterium having L-alanine-producing ability, wherein alanine dehydrogenase activity is introduced, wherein the alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari.

2. The microorganism according to claim 1, wherein the alanine dehydrogenase is encoded by an aldl, ald2, or alaD gene.

3. The microorganism according to claim 1, wherein 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.

4. The microorganism according to claim 1, wherein the gene encoding the alanine dehydrogenase comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:

8.

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

6. The microorganism according to any one of claims 1 to 5, wherein the microorganism of the genus Corynebacterium has increased L-alanine-producing ability compared to an unmodified microorganism.

7. A method of producing L-alanine comprising: a microorganism of the genus Corynebacterium having L-alanine-producing ability, wherein alanine dehydrogenase activity is introduced, wherein the alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari.

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

9. A composition for producing L-alanine, comprising: a microorganism of the genus Corynebacterium having L-alanine-producing ability, wherein alanine dehydrogenase activity is introduced, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, wherein the alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari.

10. Use of a microorganism of the genus Corynebacterium, wherein alanine dehydrogenase activity is introduced, wherein the alanine dehydrogenase is derived from Bacillus licheniformis, Bacillus amyloliquefaciens, or Laceyella sacchari.

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