Variants of aspartate 1-decarboxylase derived from tribolium castaneum and microorganisms comprising same

Abstract

The present application provides: a variant polypeptide having aspartate 1-decarboxylase activity; a microorganism comprising the variant polypeptide; a composition for producing a beta-alanine or a beta-alanine derivative compound, said composition comprising said microorganism; and a method for producing beta-alanine or a beta-alanine derivative compound, the method comprising a step of culturing the microorganism.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0034233 filed on March 18, 2022, and all contents disclosed in the corresponding Korean patent application document are incorporated as a part of this specification.

[0003] The present invention provides a variant polypeptide of aspartate 1-decarboxylase derived from red flour beetle (T. castaneum); a microorganism comprising the variant polypeptide; a composition for producing β-alanine or a β-alanine derivative compound, the composition comprising the microorganism; and a method for producing β-alanine or a β-alanine derivative compound, the method comprising the step of culturing the microorganism. Background Art

[0004] β-alanine is a naturally occurring β-amino acid in which the amino group is bound to the β carbon. β-alanine is naturally present in foods such as poultry, meat and fish and is used to synthesize carnosine in muscle.

[0005] β-Alanine-derived compounds, such as β-alanine and pantothenic acid, are one of the commercially important substances and are used in various industrial fields, such as health foods, foods, pharmaceuticals, animal feeds, etc.

[0006] Therefore, there is a need to develop a microorganism having an advantageous effect in the biotechnological preparation of β-alanine and / or β-alanine-derived compounds, and a technology for preparing β-alanine and / or β-alanine-derived compounds using the microorganism.

[0007] Prior art

[0008] Patent Literature

[0009] (Patent Document 1) U.S. Patent No. 7718205 Summary of the invention

[0010] Technical issues

[0011] The present application provides a variant polypeptide having aspartate 1-decarboxylase activity, wherein the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

[0012] The present application provides a polynucleotide encoding a variant polypeptide.

[0013] The present application provides a recombinant vector comprising the polynucleotide.

[0014] The present application provides a microorganism comprising a variant polypeptide and / or a polynucleotide encoding the variant polypeptide.

[0015] The present application provides a composition for producing β-alanine or β-alanine derivative compounds, which comprises a microorganism comprising a variant polypeptide and / or a polynucleotide encoding the variant polypeptide.

[0016] The present application provides a method for producing β-alanine or a β-alanine derivative compound, comprising culturing a microorganism comprising a variant polypeptide and / or a polynucleotide encoding the variant polypeptide.

[0017] The present application provides a use of a microorganism for producing β-alanine and / or β-alanine-derived compounds.

[0018] The present application provides a use of a microorganism for preparing a composition for producing β-alanine and / or β-alanine derivative compounds.

[0019] Technical Solution

[0020] In the present application, a variant polypeptide having aspartate 1-decarboxylase (or PanD protein) activity capable of improving the production capacity of β-alanine and / or β-alanine-derived compounds, or a recombinant microorganism (strain) having excellent β-alanine and / or β-alanine-derived compound production capacity into which the variant polypeptide is introduced is provided.

[0021] In one aspect, the present application can provide a variant polypeptide having aspartate 1-decarboxylase activity to achieve the above object. The variant polypeptide can include replacing the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 with another amino acid.

[0022] Another embodiment provides a polynucleotide encoding (or coding) the polypeptide.

[0023] Other embodiments provide a recombinant vector comprising a polynucleotide. The recombinant vector can be used as an expression vector for a polypeptide.

[0024] Other embodiments provide a microorganism comprising at least one selected from the group consisting of a polypeptide, a polynucleotide encoding the polypeptide, and a recombinant vector comprising the polynucleotide. The microorganism may be a microorganism that produces β-alanine and / or β-alanine-derived compounds.

[0025] Other embodiments provide a composition for producing β-alanine and / or β-alanine-derived compounds, comprising the microorganism.

[0026] Other embodiments provide a method for producing β-alanine and / or β-alanine derivative compounds, comprising culturing a microorganism.

[0027] Hereinafter, it will be described in more detail.

[0028] Peptides

[0029] In one aspect of the present application, a variant polypeptide having aspartate 1-decarboxylase (or PanD protein) activity is provided. The variant polypeptide having aspartate 1-decarboxylase activity may be a variant polypeptide in which at least one amino acid residue in the aspartate 1-decarboxylase protein is substituted, deleted or inserted.

[0030] The protein introduced with mutations of the present application may be a protein having aspartate 1-decarboxylase activity. The sequence of the protein may be obtained from the known database NCBI, and specifically, it may be composed of the amino acid sequence of SEQ ID NO: 51, or may contain the amino acid sequence of SEQ ID NO: 51 and have the activity of aspartate 1-decarboxylase, but is not limited thereto. In other words, it is not excluded to add meaningless sequences before and after the amino acid sequence of SEQ ID NO: 51, or naturally occurring mutations, or silent mutations thereof, and as long as it has the same or corresponding activity as the protein containing the amino acid sequence of SEQ ID NO: 51, it may correspond to the protein introduced with mutations of the present application. For example, the protein introduced with mutations of the present application may be a protein composed of the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity thereto. Furthermore, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein, proteins having an amino acid sequence in which some sequences are deleted, modified, substituted or added are also included in the scope of the protein introduced with mutations of the present application.

[0031] In one embodiment, the variant polypeptide having aspartate 1-decarboxylase activity can be a polypeptide in which the amino acid at residue 139 of the amino acid sequence corresponding to SEQ ID NO: 51 is substituted by another amino acid, wherein the other amino acid is an amino acid selected from the group consisting of tryptophan (Trp, W), histidine (His, H), tyrosine (Tyr, T), alanine (Ala, A), cysteine ​​(Cys, C), proline (Pro, P), serine (Ser, S), leucine (Leu, L), isoleucine (Ile, I), arginine (Arg, R), lysine (Lys, K), valine (Val, V), methionine (Met, M), aspartic acid (Asp, D), glutamic acid (Glu, E), glycine (Gly, G), asparagine (Asn, N), glutamine (Gln, Q) and tyrosine (Tyr, Y), and is different from the original amino acid (phenylalanine Phe, F). In a specific embodiment, the variant polypeptide having aspartate 1-decarboxylase activity may be a polypeptide in which the amino acid at the 139th residue of the amino acid sequence corresponding to SEQ ID NO: 51 is substituted by a basic amino acid (arginine, histidine or lysine), an acidic amino acid (aspartic acid or glutamic acid), a polar amino acid (serine, threonine, asparagine or glutamine), alanine, valine, isoleucine, leucine, tyrosine or glycine. In a specific embodiment, the variant polypeptide having aspartate 1-decarboxylase activity may be a polypeptide in which the amino acid at the 139th residue of the amino acid sequence corresponding to SEQ ID NO: 51 is substituted by threonine, alanine, serine, leucine, isoleucine, valine, aspartic acid, glycine, histidine or tyrosine. In a specific embodiment, the variant polypeptide having aspartate 1-decarboxylase activity may be a polypeptide in which the amino acid at the 139th residue of the amino acid sequence corresponding to SEQ ID NO: 51 is substituted by tyrosine. In a specific embodiment, the variant of aspartate 1-decarboxylase may consist of at least one amino acid sequence selected from SEQ ID NO: 52 to SEQ ID NO: 61, or comprise at least one amino acid sequence selected from SEQ ID NO: 52 to SEQ ID NO: 61.

[0032] Compared to a wild-type polypeptide having aspartate 1-decarboxylase activity, the variant polypeptide of the present application may have the property of increasing the production capacity of β-alanine and / or β-alanine-derived compounds.

[0033] Obviously, even if some amino acid sequences other than the amino acid corresponding to the 139th amino acid residue of the amino acid sequence of SEQ ID NO: 51 are deleted, modified, substituted or added in the variant polypeptide, as long as it exhibits the activity of aspartate 1-decarboxylase, it is also included in the variant polypeptide of the present application.

[0034] For example, addition or deletion of sequences at the N-terminus, C-terminus and / or within the amino acid sequence that do not change the function of the variant polypeptide of the present application, possible naturally occurring mutations, silent mutations or conservative substitutions.

[0035] A "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0036] In other words, in the variant polypeptide of the present application, the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 can be substituted by another amino acid, and the variant polypeptide of the present application can have or comprise an amino acid sequence having the following homology or identity with the amino acid sequence of SEQ ID NO: 51: 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, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.3% or more, 99.5% or more, 99.7% or more, or 99.9% or more. The amino acid sequence of IDNO:51 has 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, 98.2% or more, 98.4% or more, 98.6% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.3% or more, 99.5% or more, 99.7% or more, or 99.9% or more homology or identity, but is not limited thereto. In addition, in one embodiment, the variant polypeptide of the present application may have or comprise the following sequence, in an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with SEQ ID NO:51, the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 is substituted by another amino acid; or the variant polypeptide of the present application consists of the following sequence, or essentially consists of the following sequence: in an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with SEQ ID NO:51, the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 is substituted by another amino acid, but is not limited thereto.

[0037] In one embodiment, the variant polypeptide may have 70% or more and less than 100%, 75% or more and less than 100%, 80% or more and less than 100%, 85% or more and less than 100%, 90% or more and less than 100%, 95% or more and less than 100%, 96% or more and less than 100%, 97% or more and less than 100%, 98% or more and less than 100%, 98.2% or more and less than 100%, 98.4% or more and less than 100%, 98.6% or more and less than 100%, 98.8% or more and less than 100%, 98.9% or more and less than 100%, 99.1% or more and less than 100%, 99.2% or more and less than 100%, 99.3% or more and less than 100%, 99.4% or more and less than 100%, 99.6% or more and less than 100%, 99.8% or more and less than 100%, 99.9% or more and less than 100%. % or more and less than 100%, 99% or more and less than 100%, 99.1% or more and less than 100%, 99.3% or more and less than 100%, 99.5% or more and less than 100%, 99.77% or more and less than 100%, 99.3% or more and less than 100%, 99.5% or more and less than 100%, 99.7% or more and less than 100%, 99.9% or more and less than 100% sequence homology or sequence identity, or comprises the sequence, or consists of the sequence, or consists essentially of the sequence, but is not limited thereto.

[0038] In the present application, the term "variant polypeptide" refers to a polypeptide that differs from the amino acid sequence before the variant polypeptide mutation by conservative substitution and / or modification of at least one amino acid, but retains the function or property.

[0039] The variant polypeptide can generally be identified by modifying at least one amino acid of the amino acid sequence of the polypeptide and evaluating the characteristics of the modified polypeptide. In other words, the ability of the variant polypeptide can be increased, unchanged or decreased compared to the polypeptide before mutation. In addition, some variant polypeptides may include variant polypeptides in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, is removed. Another variant polypeptide may include a variant polypeptide in which a portion is removed from the N- and / or C-terminus of the mature protein. The term "variant polypeptide" can be used interchangeably with terms such as mutant polypeptide, modified polypeptide, variant polypeptide, mutant protein, mutation and variant (such as English expressions modification, modified polypeptide, modified protein, mutant, mutein, divergent, variant, etc.), as long as it is a term used in the sense of mutation, without limitation thereto. For the purpose of the present application, a variant polypeptide may be a polypeptide comprising the following amino acid sequence, in which the amino acid corresponding to the 139th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

[0040] In addition, variant polypeptides may include amino acid deletions or additions that have minimal effects on the properties and secondary structure of the polypeptide. For example, at the N-terminus of a variant polypeptide, a signal (or leader) sequence that participates in co-translation or post-translational protein transport may be combined. In addition, variant polypeptides may be combined with another sequence or joint to achieve confirmation, purification or synthesis.

[0041] Polynucleotide

[0042] Another aspect provides a polynucleotide encoding a variant polypeptide.

[0043] In the present application, the term "polynucleotide" refers to a DNA or RNA chain of a specific length or longer, which is a nucleotide polymer in which nucleotide monomers are linked by covalent bonds to form a long chain.

[0044] The polynucleotide of the present application may encode a variant polypeptide in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid. In addition, the polynucleotide of the present application may comprise a base sequence encoding a variant polypeptide in which the codons corresponding to the 415th to 417th residues in the nucleotide sequence of SEQ ID NO: 62 are substituted by codons encoding other amino acids. As one embodiment of the present application, the polynucleotide of the present application may comprise a base sequence encoding an amino acid sequence described in any one of SEQ ID NOs selected from SEQ ID NO: 52 to SEQ ID NO: 61. As one embodiment of the present application, the polynucleotide of the present application may have or comprise any one of the sequences selected from SEQ ID NO: 63 to SEQ ID NO: 72. In addition, the polynucleotide of the present application may consist of or be substantially composed of any one of the sequences selected from SEQ ID NO: 63 to SEQ ID NO: 72.

[0045] Taking into account the codon degeneracy or preferred codons in an organism intended to express the variant polypeptide of the present application, the polynucleotide of the present application may have various modifications in the coding region within the range of not changing the amino acid sequence of the variant polypeptide of the present application. Specifically, the polynucleotide of the present application may have or comprise a base sequence having the following homology or identity with any one of the sequences selected from SEQ ID NO: 63 to SEQ ID NO: 72: 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.99% or more. Any of the sequences in NO:72 has 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity, but is not limited thereto.

[0046] In one embodiment, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 139 in SEQ ID NO: 51 may be one of the codons encoding tryptophan, histidine, tyrosine, alanine, cysteine, proline, serine, leucine, isoleucine, arginine, lysine, valine, methionine, aspartic acid, glutamic acid, glycine, asparagine, glutamine or tyrosine. In a specific embodiment, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 139 in SEQ ID NO: 51 may be one of the codons encoding a basic amino acid (arginine, histidine or lysine), an acidic amino acid (aspartic acid or glutamic acid), a polar amino acid (serine, threonine, asparagine or glutamine), alanine, valine, isoleucine, leucine, tyrosine or glycine. In a specific embodiment, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 139 in SEQ ID NO: 51 may be one of the codons encoding threonine, alanine, serine, leucine, isoleucine, valine, aspartic acid, glycine, histidine or tyrosine. In a specific embodiment, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 139 in SEQ ID NO: 51 may be one of the codons encoding tyrosine.

[0047] In addition, the polynucleotide of the present application may include without limitation, as long as it can be a probe prepared from a known gene sequence, such as a sequence that can hybridize with all or part of a sequence complementary to the polynucleotide sequence of the present application under stringent conditions. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. The conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides having high homology or identity to each other (polynucleotides having 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more) can be listed. The conditions in which polynucleotides with 9.9% or more homology or identity to each other are hybridized, while polynucleotides with lower homology or identity to each other than the polynucleotides do not hybridize, or the conditions in which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions for common Southern hybridization.

[0048] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches may occur between bases 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, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0049] Specifically, hybridization conditions including a hybridization step at a Tm value of 55°C can be used, and the above conditions can be used to detect polynucleotides having homology or identity with the polynucleotides of the present application. In addition, the Tm 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 the purpose.

[0050] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and variables are well known in the art (eg, J. Sambrook et al., supra).

[0051] In the present specification, a polynucleotide (which can be used interchangeably with "gene") or a polypeptide (which can be used interchangeably with "protein") "comprising a specific nucleic acid sequence or amino acid sequence or consisting of a specific nucleic acid sequence or amino acid sequence or expressing a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide basically includes the specific nucleic acid sequence or amino acid sequence, and it may be interpreted as including "substantially equivalent sequences" in which meaningless mutations (deletions, substitutions, modifications and / or additions) are applied to the specific nucleic acid sequence or amino acid sequence within the scope of maintaining the original function and / or desired function of the polynucleotide or polypeptide (or not excluding meaningless mutations).

[0052] In this specification, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or base sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0053] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard array algorithms, and can be used together with the default gap penalty set up by the program used. Basically, sequences of homology or identity can hybridize with the whole or part of the sequence under medium or high stringency conditions usually. Obviously, hybridization also includes hybridization of polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotides.

[0054] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program, such as using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. In addition, the Needlean-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of 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) (including the 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, BLAST or ClustalW from the National Center for Biotechnology Information Database can be used to determine homology, similarity or identity.

[0055] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48: 443, as known in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program can be defined as the value obtained by dividing the total number of symbols in the shorter of the two sequences by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a binary comparison matrix (containing identical values ​​of 1 and different values ​​of 0) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol of 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] In this specification, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in a polypeptide. Confirming an amino acid at a corresponding position can be determining a specific amino acid in a sequence with reference to a specific sequence. "Corresponding region" as used in this application generally refers to a similar or corresponding position in a related protein or a reference protein.

[0057] For example, any amino acid sequence is aligned with SEQ ID NO: 52, and based thereon, each amino acid residue in the amino acid sequence can be numbered by reference to the numbered position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 52. For example, the sequence alignment algorithms described in this application can compare with a query sequence (also referred to as a "reference sequence") to confirm the position of an amino acid, or the position at which a modification such as a substitution, insertion or deletion has occurred.

[0058] For this alignment, for example, the Needlean-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0059] In this specification, the term "enhancing" of polypeptide activity refers to that the activity of polypeptide increases compared with endogenous activity.Enhancing can be used interchangeably with terms such as activation, up-regulation, overexpression, increase.Herein, activation, up-regulation, overexpression and increase can include showing an activity that does not originally exist, or showing an activity that is improved compared with the activity before endogenous activity or modification."Endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain or unmodified microorganism before the property change when the genetic mutation caused by natural or artificial factors changes.This can be used interchangeably with "activity before modification".Compared with endogenous activity, polypeptide activity "enhancing", "up-regulation", "overexpression" or "increase" refers to the activity and / or concentration (expression level) of a specific polypeptide originally possessed by a parent strain or unmodified microorganism before the property change, and polypeptide activity improves.

[0060] Enhancement can be achieved by introducing exogenous polypeptides or can be to enhance the activity and / or concentration (expression level) of endogenous polypeptides. Whether the polypeptide activity is enhanced can be confirmed by the increase in the degree of activity, the expression level of the corresponding polypeptide or the amount of product released from the corresponding polypeptide.

[0061] For the enhancement of polypeptide activity, various methods known in the art can be applied, and there is no restriction, as long as the activity of the target polypeptide can be enhanced compared with the activity of the microorganism before modification. Specifically, genetic engineering and / or protein engineering known to those skilled in the art can be used, which are conventional methods of molecular biology, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0062] Specifically, the enhancement of the polypeptide of the present application can be:

[0063] 1) The cell copy number of the polynucleotide encoding the polypeptide increases;

[0064] 2) Replace the gene expression regulatory region encoding the polypeptide on the chromosome with a sequence with strong activity;

[0065] 3) modifying the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide;

[0066] 4) Modify the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

[0067] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide of a polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

[0068] 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding a polypeptide;

[0069] 7) codon-optimized polynucleotides encoding polypeptides;

[0070] 8) by analyzing the tertiary structure of the polypeptide and selecting modification or chemical modification of the exposed sites; or

[0071] 9) A combination of at least two selected from the above 1) to 8), but not limited thereto.

[0072] More specifically, the increase in the number of copies of the polynucleotide encoding the polypeptide in the above 1) can be achieved by introducing a vector, which can replicate and function independently of the host, wherein the polynucleotide encoding the corresponding polypeptide is operably connected. In addition, it can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out 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.

[0073] In the above 2), replacing the gene expression control region (or expression control sequence) encoding the polypeptide on the chromosome with a sequence having strong activity can be, for example, a mutation of the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, to further enhance the activity of the expression control region, or replace it with a sequence having stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, etc. As an example, the original promoter can be replaced with a strong promoter, but is not limited thereto.

[0074] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 1,058,4338 B2), O2 promoter (U.S. Patent No. 1,027,3491 B2), tkt promoter, yccA promoter, etc., but are not limited to these.

[0075] In the above 3), the modification of the start codon or 5'-UTR base sequence of the gene transcriptome encoding the polypeptide may be, for example, replacement with a base sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0076] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide is replaced with an improved amino acid sequence or polynucleotide sequence with stronger activity, thereby enhancing the activity of the polypeptide, but is not limited thereto. The polynucleotide can be specifically inserted into the chromosome for replacement by homologous recombination, but is not limited thereto. The vector used then can also include a selection marker for confirming chromosome insertion. The selection marker is as described above.

[0077] In the above 6), the introduction of an exogenous polypeptide or coding exhibiting polypeptide activity may be the introduction of an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The source or sequence of the exogenous polynucleotide is not limited, as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be carried out by those skilled in the art by appropriately selecting a known transformation method, and by expressing the introduced polynucleotide in a host cell, a polypeptide can be produced and its activity can be increased.

[0078] The codon-optimized polypeptide encoding polynucleotide in the above 7) may be codon-optimized so as to increase the transcription or translation of an endogenous polynucleotide in a host cell, or codon-optimized so as to optimize the transcription and translation of an exogenous polynucleotide in a host cell.

[0079] In the above 8), by analyzing the tertiary structure of the polypeptide and selecting the modification or chemical modification of the exposed site, the template protein candidate can be determined according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed, based on which the structure is determined and the exposed site to be modified or chemically modified is selected, and it is transformed or modified.

[0080] Such enhancement of polypeptide activity can be based on the activity or concentration of the polypeptide expressed in the wild-type or modified microbial strain, an increase in the activity or concentration expression level of the corresponding polypeptide, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0081] Recombinant vector

[0082] Other embodiments provide a recombinant vector comprising a polynucleotide. The recombinant vector can be used as an expression vector for a polypeptide. The recombinant vector can be used to insert the polynucleotide into the genome of a host cell or replace the corresponding gene of the host cell genome.

[0083] The vector of the present application may include a DNA preparation, the DNA preparation includes a base sequence of a polynucleotide encoding a target polypeptide, the base sequence is operably connected to a suitable expression control region (or expression control sequence) so that the target polypeptide is expressed in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription and translation termination. After the vector is transformed into a suitable host cell, it can be replicated or operated independently of the host genome and integrated into the genome itself.

[0084] The vector used in the present application is not particularly limited, and any vector known in the art can be used. The example of a common vector may include a plasmid, a cosmid, a virus and a phage in a natural state or a recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A etc. can be used as a phage vector or a cosmid vector, and pDZ, pBR, pUC, pBluescript II, pGEM, pTZ, pCL and pET etc. can be used as a plasmid vector. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pTop33ori vectors etc. can be used.

[0085] As an example, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for inserting the chromosome into the cell. The polynucleotide insertion into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker can be further included to confirm whether the chromosome is inserted. The selection marker is used to select the cell transformed by the vector, that is, to confirm whether the target nucleic acid molecule is inserted, and a marker that can confer a selectable phenotype such as drug resistance, auxotrophy, cytotoxic agent resistance or expression of surface polypeptide can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so transformed cells can be selected.

[0086] In the present disclosure, the term "conversion" means that a vector comprising a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism to express a polypeptide encoded by the polynucleotide in the host cell. The converted polynucleotides may include all polynucleotides, regardless of whether they are inserted into and located in the chromosome of the host cell or located outside the chromosome, as long as the converted polynucleotides can be expressed in the host cell. In addition, the polynucleotides include DNA and / or RNA encoding the target polypeptide. The polynucleotides may be introduced in any form, as long as they can be introduced into the host cell and expressed. For example, the polynucleotides may be introduced into the host cell in the form of an expression cassette, which is a gene structure comprising all elements required for self-expression. The expression cassette may generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotides may be introduced into the host cell in the form of themselves, and may be operably connected to a sequence required for expression in the host cell, but is not limited thereto.

[0087] In addition, in the above content, the term "operably linked" means that a promoter sequence capable of initiating and mediating transcription of a polynucleotide encoding a target variant of the present application is functionally linked to the polynucleotide sequence.

[0088] microorganism

[0089] Other aspects provide a microorganism comprising at least one (e.g., 1 or more, 2 or more, or 1, 2, or 3) selected from the group consisting of a variant polypeptide, a polynucleotide encoding (or encoding) a polypeptide, and a recombinant vector comprising the polynucleotide. In one embodiment, the microorganism may be a microorganism in which the production capacity of β-alanine and / or β-alanine-derived compounds is increased (or enhanced).

[0090] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms, naturally or artificially genetically modified microorganisms, and the microorganism may be a genetically modified microorganism that produces a target polypeptide, protein or product (e.g., aspartate 1-decarboxylase), wherein a specific mechanism is weakened or strengthened due to, for example, the insertion of exogenous genes and / or the enhancement or inactivation of the activity of endogenous genes.

[0091] The microorganism of the present application may be a microorganism (eg, a recombinant microorganism) genetically modified by a vector comprising a variant polypeptide or a polynucleotide encoding the variant polypeptide, but is not limited thereto. The vector is as described above.

[0092] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having the ability to produce β-alanine and / or β-alanine derivative compounds or having improved production ability (or yield) of β-alanine and / or β-alanine derivative compounds.

[0093] The microorganism of the present application may be a microorganism that naturally has the ability to produce β-alanine and / or β-alanine derivative compounds, or a microorganism that imparts or improves the ability to produce β-alanine and / or β-alanine derivative compounds compared to a microorganism that does not have the ability to produce β-alanine and / or β-alanine derivative compounds, but is not limited thereto. The microorganism that imparts or improves the ability to produce β-alanine and / or β-alanine derivative compounds may be a microorganism into which a variant polypeptide having aspartate 1-decarboxylase activity is introduced.

[0094] The microorganism (or strain, recombinant cell) has an increased (or improved) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds, or has the production capacity of β-alanine and / or β-alanine-derived compounds, which may mean that the microorganism (or strain, recombinant cell) may have an increased production capacity of β-alanine and / or β-alanine-derived compounds compared to an unmodified microorganism, a cell before recombination, a parent strain, a wild-type strain, or a microorganism into which an aspartate 1-decarboxylase derived from other microorganisms (e.g., a wild-type Tribolium castaneum) is introduced; or the microorganism is endowed with a production capacity of β-alanine or β-alanine-derived compounds different from that of an unmodified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain that does not have the production capacity of β-alanine and / or β-alanine-derived compounds.

[0095] The microorganism of the present application may be any one or more microorganisms comprising the variant polypeptide of the present application, the polynucleotide encoding the variant polypeptide of the present application, and the vector comprising the polynucleotide of the present application; a microorganism modified to express the variant polypeptide of the present application or the polynucleotide of the present application; a microorganism (such as a recombinant strain) expressing the variant of the present application or the polynucleotide of the present application; or a microorganism (such as a recombinant strain) having the activity of the variant polypeptide of the present application, but is not limited thereto.

[0096] In one embodiment, the microorganism of the present application may be a microorganism of the genus Corynebacterium. The Corynebacterium microorganism may be at least one selected from the group consisting of 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 and Corynebacterium flavescens.

[0097] Compared with the unmodified microorganism of the same kind, microorganism can have the production capacity of (increased) β-alanine and / or β-alanine derivative compounds that improve.In the present application, the term "unmodified microorganism" does not exclude the strain comprising the mutation that can be naturally present in the microorganism, and can refer to the wild-type strain or natural strain itself, or the strain before the gene mutation caused by natural or artificial factors changes the character.For example, according to one embodiment, unmodified microorganism can refer to the strain wherein not introducing the variant polypeptide of the present application or not introducing the polynucleotide encoding the variant polypeptide or before it is introduced.In addition, "unmodified microorganism" can refer to the strain introducing the aspartate 1-decarboxylase derived from the wild-type red castaneum or the polynucleotide encoding it. "Unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism" or "reference microorganism". In one embodiment, the unmodified microorganism is a test strain for comparing whether the production capacity of β-alanine and / or β-alanine-derived compounds is increased, and can specifically be a wild-type Corynebacterium glutamicum ATCC13032 strain, or a strain into which panD of a wild-type Tribolium castaneum is introduced instead of panD of a wild-type Corynebacterium glutamicum ATCC13032 strain, but is not limited thereto.

[0098] The microorganism (or strain, recombinant cell) may include mutations to further increase the production of β-alanine and / or β-alanine derivative compounds, and the position of the mutation and the position of any kind of gene and / or protein subjected to mutation may include, but are not limited to, as long as it increases the production of β-alanine and / or β-alanine derivative compounds. The recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0099] In a specific embodiment, the microorganism may be deposited as KCCM13077P.

[0100] The β-alanine derivative compound can be at least one selected from the group consisting of β-nitropropionic acid, β-amino-propionitrile, N-acetyl-β-alanine, L-aspartic acid, anserine, spermine, carnosine, β-alanylarginine, quinolinate, pantothenate (or pantothenic acid), malonic semialdehyde, malonate, acetylene-monocarboxylate, etc., but is not limited thereto.

[0101] In other embodiments, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine derivative compounds may have an increase in the production capacity (or yield) of β-alanine and / or β-alanine derivative compounds by about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, or about 150% or more, compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism is introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum is introduced, but is not limited thereto.

[0102] In other embodiments, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine derivative compounds may have an increase in production capacity (or yield) of β-alanine and / or β-alanine derivative compounds by about 2.0 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, or about 2.5 times or more, compared to the parent strain before mutation, an unmodified microorganism, a microorganism introduced with an aspartate 1-decarboxylase derived from another microorganism, or a microorganism introduced with an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum, but is not limited thereto.

[0103] In another embodiment, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase in the production capacity (or yield) of β-alanine and / or β-alanine-derived compounds by about 1.0 g / L or more, about 1.1 g / L or more, about 1.2 g / L or more, about 1.3 g / L or more, about 1.4 g / L or more, or about 1.5 g / L or more, compared to the parent strain before mutation, an unmodified microorganism, a microorganism introduced with an aspartate 1-decarboxylase derived from another microorganism, or a microorganism introduced with an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum, but is not limited thereto.

[0104] More specifically, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine derivative compounds may have an increase of about 100%, about 110%, about 120%, about 130%, about 140% or about 150% (or about 2.0 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times or about 2.5 times; or about 1.0 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L or about 1.5 g / L) in production capacity (or yield) of β-alanine and / or β-alanine derivative compounds, as compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism is introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum is introduced, but is not limited thereto.

[0105] In another embodiment, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, about 200% or more, about 210% or more, about 220% or more, about 230% or more, about 240% 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 330% or more, about 340% or more, about 350% or more, about 360% or more, about 370% or more, about 380% or more, about 390% or more, about 400% or more, about 410% or more, about 420% or more, about 430% or more, about 440% or more, about 450% or more, about 460% or more, about 470% or more, about 480% or more, about 490% or more, about 500% % or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 110% or more, about 120% or more, 125% or more, about 130% or more, about 140% or more, or about 150% or more of the production capacity (or yield) of β-alanine and / or β-alanine-derived compounds (e.g., pantothenic acid), but are not limited thereto.

[0106] In another embodiment, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase of about 1.05 times or more, about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, about 1.3 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2.0 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, about 2.5 times or more, about 2.6 times or more, about 2.7 times or more, about 2.8 times or more, about 2.9 times or more, about 3.4 times or more, about 3.5 times or more, about 3.6 times or more, about 3.7 times or more, about 3.8 times or more, about 3.9 times or more, about 4.5 times or more, about 5.6 times or more, about 5.7 times or more, about 5.8 times or more, about 5.9 times or more, about 6.7 times or more, about 6.8 times or more, about 7.9 times or more, about 8. .3 times or more, about 1.35 times or more, about 1.4 times or more, about 1.45 times or more, about 1.5 times or more, about 1.55 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2 times or more, about 2.1 times or more, about 2.2 times or more, about 2.25 times or more, about 2.3 times or more, about 2.4 times or more, or about 2.5 times or more of the production capacity (or output) of β-alanine and / or β-alanine derivative compounds, but not limited to this.

[0107] In another embodiment, the microorganism (or strain, recombinant cell) having improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase of about 0.05 g / L or more, about 0.06 g / L or more, about 0.07 g / L or more, about 0.08 g / L or more, about 0.09 g / L or more, about 0.1 g / L or more, about 0.13 g / L or more, about 0.16 g / L or more, about 0.19 g / L or more, about 0.2 g / L or more, about 0.23 g / L or more, about 0.26 g / L or more, about 0.27 g / L or more, about 0.28 g / L or more, about 0.29 g / L or more, about 0.30 g / L or more, about 0.31 g / L or more, about 0.32 g / L or more, about 0.33 g / L or more, about 0.36 g / L or more, about 0.37 g / L or more, about 0.38 g / L or more, about 0.39 g / L or more, about 0.40 g / L or more, about 0.41 g / L or more, about 0.42 g / L or more, about 0.43 g / L or more, about 0.44 g / L or more, about 0.45 g / L or more, about 0.46 g / L or more, about 0.47 g / L or more, about 0.48 g / L or more, about 0.49 g / L or more, about 0.50 g / L or more, about 0.51 g / L or more, about 0.52 g / L or more, About 0.29 g / L or more, about 0.3 g / L or more, about 0.33 g / L or more, about 0.36 g / L or more, about 0.39 g / L or more, about 0.4 g / L or more, about 0.43 g / L or more, about 0.46 g / L or more, about 0.49 g / L or more, about 0.5 g / L or more, about 0.53 g / L or more, about 0.56 g / L or more , about 0.59 g / L or more, about 0.6 g / L or more, about 0.63 g / L or more, about 0.67 g / L or more, about 0.69 g / L or more, about 0.7 g / L or more, about 0.8 g / L or more, about 0.9 g / L or more, or about 1.0 g / L or more of the production capacity (or yield) of β-alanine and / or β-alanine derivative compounds, but not limited to this.

[0108] More specifically, the microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase of about 10%, about 15%, about 20%, about 25%, about 35%, about 40%, about 55%, about 100% or about 150% compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism is introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum is introduced. 25% (or about 1.1 times, about 1.15 times, about 1.2 times, about 1.25 times, about 1.35 times, about 1.4 times, about 1.55 times, about 2 times or 2.25 times; or about 0.06 g / L, about 0.1 g / L, about 0.13 g / L, about 0.16 g / L, about 0.23 g / L, about 0.26 g / L, about 0.36 g / L, about 0.5 g / L or about 0.6 g / L) of the production capacity (or yield) of β-alanine and / or β-alanine derivative compounds, but not limited to this.

[0109] The term "about" includes all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within the range equivalent to or similar to the value after the term about, but is not limited thereto.

[0110] Other aspects provide a composition for producing β-alanine and / or β-alanine derivative compounds, which comprises the microorganism of the present application, a culture medium in which the microorganism is cultured, or a combination thereof.

[0111] The composition of the present application may also contain any suitable excipients commonly used in the production of β-alanine and / or β-alanine derivative compound compositions, and the excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or tonicity agents, etc., but are not limited thereto.

[0112] In the composition of the present application, the microorganism (strain) or culture medium etc. are as described in other aspects above.

[0113] Another aspect provides a method for producing β-alanine and / or β-alanine derivative compounds, which comprises culturing the microorganism of the present application in a culture medium.

[0114] The method for producing β-alanine and / or β-alanine derivative compounds of the present application may include culturing the microorganism of the present application in a culture medium. The microorganism of the present application is as described above.

[0115] In the present application, "cultivation" refers to growing the microorganisms comprising the variant polypeptides of the present application under appropriately adjusted environmental conditions. The cultivation process of the present application can be carried out according to appropriate culture media and culture conditions known in the art. Depending on the selected strain, those skilled in the art can easily adjust and use the cultivation process. Specifically, the cultivation can be batch, continuous and / or batch fed, but is not limited thereto.

[0116] In the present application, "culture medium" refers to a material that mixes the nutrients required for culturing the microorganism of the present application as the main component, and provides nutrients and growth factors, etc., including water that is essential for survival and growth. Specifically, the culture medium and other culture conditions for culturing the microorganism of the present application can be used, as long as it is a culture medium for culturing common microorganisms without particular limitation, but the microorganism of the present application can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions by adjusting temperature, pH, etc.

[0117] Specifically, the culture medium of the microorganisms used in the present application, for example, the culture medium of Corynebacterium strains can be found in the document ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0118] In the present application, as a carbon source, it can include: carbohydrates such as glucose, sucrose, 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, natural organic nutrients such as starch hydrates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residues and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted with reducing sugars) can be used, and other carbon sources of appropriate amounts can be used differently without limitation. These carbon sources can be used alone or in combination of at least two, but are not limited thereto.

[0119] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium citrate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., organic nitrogen sources such as amino acids including glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrate, fish or its degradation product, defatted soybean cake or its degradation product, etc. can be used alone or in combination of at least two, but are not limited thereto.

[0120] As a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or a sodium salt corresponding thereto may be included. As an inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition to these, amino acids, vitamins and / or appropriate precursors may be included. These components or precursors may be added in batches or in a continuous manner. However, this is not limited thereto.

[0121] In addition, by appropriate method during the cultivation of the microorganism of the present application, in substratum, add compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid etc., regulate the pH of substratum.In addition, during the cultivation, defoamer such as fatty acid polyglycol ester can be used to suppress foaming.In addition, in order to maintain the aerobic state of substratum, oxygen or oxygen-containing gas can be injected into substratum, or in order to keep anaerobic and micro-aerobic state, gas can not be injected or nitrogen, hydrogen or carbon dioxide can be injected, but is not limited to this.

[0122] The culture temperature in the culture of the present application may be maintained at 20° C.-45° C., specifically 25° C.-40° C., and the culture may be performed for about 10-160 hours, but is not limited thereto.

[0123] The β-alanine and / or β-alanine derivative compound produced by the culture of the present application may be released into the medium or retained in the cells.

[0124] The method for producing β-alanine and / or β-alanine derivative compounds of the present application may further include, for example, preparing the microorganism (strain) of the present application, preparing a culture medium for culturing the microorganism, or a combination thereof (in any order) before culturing.

[0125] The production method of β-alanine and / or β-alanine derivative compounds of the present application may further include recovering β-alanine and / or β-alanine derivative compounds from a culture medium (a culture medium in which culture is performed) or a microorganism (e.g., a Corynebacterium strain) according to the culture. The recovery may further include after the culture.

[0126] Recovery can be based on the culture method of the microorganism of the present application, such as batch, continuous or batch feeding culture method, etc., by using appropriate methods known in the art to collect the target β-alanine and / or β-alanine derivative compounds. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out method), extraction, ultrasonic crushing, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or a combination of these methods, and using appropriate methods known in the art, the target β-alanine and / or β-alanine derivative compounds can be recovered from the culture medium or microorganism.

[0127] In addition, the production method of β-alanine and / or β-alanine derivative compounds of the present application may also include a purification step. Suitable methods known in the art may be used for purification. In one embodiment, when the method for producing β-alanine and / or β-alanine derivative compounds 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 by integrating into one step, without limitation thereto.

[0128] Other aspects provide a use of the microorganism of the present application for producing β-alanine and / or β-alanine derivative compounds or preparing a composition for producing β-alanine and / or β-alanine derivative compounds. The microorganism, β-alanine and / or β-alanine derivative compounds or the composition for producing β-alanine and / or β-alanine derivative compounds of the present application are as described above.

[0129] Beneficial Effects

[0130] The present application provides a variant polypeptide having aspartate 1-decarboxylase activity, a microorganism comprising the variant polypeptide, a composition comprising the microorganism for producing β-alanine and / or β-alanine derivative compounds, and a method for producing β-alanine and / or β-alanine derivative compounds, the method comprising culturing the microorganism, and the microorganism having excellent production capacity of β-alanine and / or β-alanine derivative compounds. DETAILED DESCRIPTION

[0131] Hereinafter, the present invention will be described in more detail by the following examples. However, they are only intended to illustrate the present invention, but the scope of the present invention is not limited by these examples.

[0132] Example 1. Construction of random mutant strains and selection of panD variant polypeptide (variant) expression strains based on NTG artificial mutation method

[0133] In this example, in order to obtain a microbial mutant strain having an improved ability to produce more β-alanine, Corynebacterium glutamicum ATCC13032 (ATCC13032ΔpanD::panD(TC)) into which the panD gene (aspartate 1-decarboxylase or PanD protein) derived from Tribolium castaneum was introduced was used as a parent strain to induce mutation.

[0134] ATCC13032ΔpanD::panD(TC) strain was constructed by the following method. First, a vector for deleting the panD gene endogenously present in Corynebacterium glutamicum ATCC13032 was constructed. PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NOs:73 and 74 and SEQ ID NOs:75 and 76. PCR was performed at 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute, repeated 30 times. As a result, a gene fragment of 1000 bp from the upper part of the panD gene and a gene fragment of 1000 bp from the lower part of the pan gene were obtained, respectively, and each amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction.

[0135] The pDCM2 (Korean Patent No. 2278000) vector was treated with the restriction endonuclease smaI and heat-treated at 65°C for 20 minutes and the DNA fragments (a 1000 bp gene fragment from the upper part of the panD gene and a 1000 bp gene fragment from the lower 1000 bp of the panD gene) were cloned using TaKaRa's Infusion cloning kit according to the manual provided so that the molar concentration (M) was 2:1:1, thereby constructing the vector pDCM2_ΔpanD for deleting the SCD gene on the chromosome.

[0136] In order to prepare the panD gene derived from red castaneum, genomic DNA extracted from red castaneum was used as a template and PCR was performed using primers 77 and 78. PCR was repeated 30 times by 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute and 30 seconds, resulting in a DNA fragment of 1640 bp. In order to obtain the lysC promoter derived from Corynebacterium glutamicum, genomic DNA of Corynebacterium glutamicum was used as a template and PCR was performed using primers 79 and 90 in the same manner as in the above-mentioned embodiment method to obtain a DNA fragment. The pDCM2_ΔpanD vector and the obtained DNA fragment, which were treated with restriction endonucleases smaI and then heat-treated at 65°C for 20 minutes, were cloned using TaKaRa's Infusion cloning kit according to the manual provided, so that the molar concentration (M) was 2:1:1 to construct the vector pDCM2_ΔpanD::pan D (TC) for introducing the pan gene derived from red castaneum into the chromosome.

[0137] By transforming the constructed vector pDCM2_ΔpanD::panD (TC) into Corynebacterium glutamicum ATCC13032 by electroporation, and by a secondary hybridization process, strains (ΔpanD::panD (TC)) with pan genes derived from Escherichia coli introduced on chromosomes were obtained respectively. The following primer combinations were used to confirm the appropriate replacement of panD derived from Escherichia coli using the MASA (mutant allele specific amplification) PCR method (Takeda et al., Hum. Mutation, 2, 112-117 (1993)). In other words, the primer combination matching Tribolium castaneum panD (SEQ ID NOs: 81 and 80 and SEQ ID NOs: 77 and 82) was first determined by selecting an amplification strain, and the panD gene sequence of the selected strain was analyzed by using the primer combination of SEQ ID NO: 81 and SEQ ID NO: 82, completing the secondary selection.

[0138] The selected Corynebacterium glutamicum ATCC13032ΔpanD::panD (TC) strain was activated by cultivating 16 hours in activation medium, and inoculated and cultivated 14 hours in the minimum medium of 121 ℃ sterilization for 15 minutes, then reclaimed e mL culture fluid. The culture fluid recovered was washed with 100mM citric acid buffer (citric buffer), then NTG (N-methyl-N'-nitro-N-nitrosoguanidine) was added, and final concentration was 200mg / L, processed 20 minutes, washed with 100mM phosphate buffer (phosphate buffer). As the result of measuring mortality rate by applying the strain processed by NTG on the minimum medium, the mortality rate was shown as 85%. Inoculate and cultivate surviving cells in production medium, finally select the mutant strain that demonstrates the most excellent beta-alanine production capacity, named after Corynebacterium glutamicum CJVB5-03-1.

[0139] The composition of the culture medium used in this embodiment is as follows:

[0140] <Activation medium>

[0141] Beef extract 1%, peptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2

[0142] <Production medium>

[0143] Glucose 10%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, iron sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 6.7 mg / L, biotin 50 μg / L, thiamine·HCl 100 μg / L, pH 7.2

[0144] <Minimum Medium>

[0145] Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium dihydrogen phosphate 0.1%, urea 0.1%, thiamine 0.001%, biotin 200μg / L, agar 2%, pH 7.2

[0146] In order to compare the β-alanine production capacity of Corynebacterium glutamicum ΔpanD::panD(TC) and the obtained mutant strain Corynebacterium glutamicum CJVB5-03-1, the Corynebacterium glutamicum ΔpanD::panD(TC) strain and the CJVB5-04 mutant strain were inoculated into 250 ml baffled flasks containing 25 ml of production medium, respectively, and then cultured with shaking at 200 rpm at 30°C for 48 hours.

[0147] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then the supernatant was diluted to 1 / 10 with TDW (triplet distilled water) and subjected to HPLC analysis to measure the concentration of β-alanine. The results are shown in Table 1 below.

[0148] [Table 1]

[0149] β-Alanine production ability of NTG-based mutant strains

[0150] β-Alanine concentration (g / L) ATCC13032ΔpanD::panD(TC) 0.5 CJVB5-03-1 1.2

[0151] The results, as shown in Table 1 above, confirmed that Corynebacterium glutamicum ATCC13032ΔpanD::panD(TC) produced β-alanine at a concentration of 0.5 g / L, and the mutant strain Corynebacterium glutamicum CJVB5-03-1 according to the present application produced β-alanine at a concentration of 1.2 g / L, and compared with the parent strain, the production capacity of β-alanine increased by about 2.4 times (increased by about 140%).

[0152] Based on the above results, as a result of genome sequencing of genes in the biosynthetic pathway in which β-alanine is synthesized from pyruvate, random substitution in the panD gene derived from Tribolium castaneum in the mutant strain Corynebacterium glutamicum CJVB5-03-1 was confirmed.

[0153] Specifically, it was confirmed that the sequence encoding the phenylalanine (F) at position 139 of the wild-type PanD protein of Tribolium castaneum was mutated to a sequence encoding tyrosine (Y). The amino acid sequence of the mutant PanD (F (phenylalanine) at position 139 mutated to Y (tyrosine)) protein derived from the wild-type PanD (SEQ ID NO: 51) of Tribolium castaneum is represented by SEQ ID NO: 52.

[0154] The above results confirmed that the mutant strain obtained by random mutagenesis can produce β-alanine with high efficiency and high yield.

[0155] Example 2. Confirmation of pantothenate-producing ability of aspartate 1-decarboxylase variant polypeptides

[0156] In Example 1, it was confirmed that the β-alanine production capacity of the variant polypeptide of aspartate 1-decarboxylase of Tribolium castaneum was increased. In addition, in order to confirm the pantothenate production capacity of the variant polypeptide having aspartate 1-decarboxylase activity of Tribolium castaneum, a microorganism with enhanced 3-methyl-2-oxobutyrate methyl hydroxymethyltransferase (PanB) activity was constructed.

[0157] First, a vector for deleting the endogenous panB gene (gene encoding 3-methyl-2-oxobutyrate methyl hydroxymethyltransferase or PanB protein) present in Corynebacterium glutamicum ATCC13032 was constructed. Genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR was performed using primers of SEQ ID NOs:41 and 42 and SEQ ID NOs:43 and 44. PCR was performed under the conditions of 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute, repeated 30 times. As a result, a gene fragment of 1,000 bp from the upper part of the panB gene and a gene fragment of 1,000 bp from the lower part of the panB gene were obtained, respectively, and each amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction.

[0158] The pDCM2 (Korean Patent No. 2278000) vector and DNA fragments (a 1,000 bp gene fragment from the upper part of the panB gene and a 1,000 bp gene fragment from the lower part of the panB gene) treated with the restriction endonuclease smaI and heat-treated at 65°C for 20 minutes were used according to the provided manual using TaKaRa's Infusion cloning kit so that the molar concentration (M) was 2:1:1, thereby constructing a vector pDCM2_ΔpanB for deleting the panB gene on the chromosome.

[0159] In order to prepare the panB gene derived from red castaneum, genomic DNA extracted from Escherichia coli K12 wild-type strain (KCTC1116) was used as a template and PCR was performed using primers 47 and 48. PCR was repeated 30 times by 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute and 30 seconds, to obtain a DNA fragment of 795bp. In order to obtain the lysC promoter derived from Corynebacterium glutamicum, genomic DNA of Corynebacterium glutamicum was used as a template and PCR was performed using primers 45 and 46 in the same manner as the above-mentioned embodiment method to obtain a DNA fragment. The pDCM2_ΔpanB vector treated with restriction endonuclease smaI and then heat-treated at 65°C for 20 minutes, and the obtained DNA fragment was cloned using TaKaRa's Infusion cloning kit according to the manual provided, so that the molar concentration (M) was 2:1:1, to construct the vector pDCM2_ΔpanB::panB (EC), which is used to introduce the panB gene derived from Escherichia coli into the chromosome.

[0160] By electroporation, the constructed vector pDMC2_ΔpanB::panB (EC) was transformed into Corynebacterium glutamicum ATCC13032ΔpanD::panD (TC) and CJVB5-03-1, respectively, and by a secondary hybridization process, strains (ΔpanB::panB (EC)) were obtained in which the panB gene derived from Escherichia coli was introduced on the chromosome. The following primer combination was used to confirm the appropriate replacement of panB derived from Escherichia coli using the MASA (mutant allele specific amplification) PCR method (Takeda et al., Hum. Mutation, 2, 112-117 (1993)). In other words, the primer combination matching the panB gene of Escherichia coli (SEQ ID No: 49 and 46 and SEQ ID No: 47 and 50) was first determined by selecting the amplified strain, and the panB gene sequence of the selected strain was analyzed by using the primer combination of SEQ ID NO: 49 and SEQ ID NO: 50.

[0161] For the constructed strains, in order to confirm the pantothenic acid production ability, the parent strain and the strain were respectively inoculated into 250 ml angle baffle flasks containing 25 ml production medium, wherein the production medium was respectively composed of the composition used as an example, and then shake-cultured at 200 rpm at 33°C for 48 hours to produce pantothenic acid. The results are shown in Table 2 below.

[0162] [Table 2]

[0163] Pantothenate production ability of NTG-based mutant strains

[0164]

[0165] The results are shown in Table 2 above, which confirms that Corynebacterium glutamicum ATCC13032ΔpanD::panD(TC)ΔpanB::panB(EC) produces pantothenic acid at a concentration of 0.4 g / L, but Corynebacterium glutamicum CJVB5-04ΔpanB::panB(EC) according to the present application produces pantothenic acid at a concentration of 0.9 g / L, and therefore, the pantothenic acid production capacity is increased by approximately 2.25 times (increased by approximately 125%) compared to the parent strain.

[0166] The above results indicate that the aspartate 1-decarboxylase variant polypeptide derived from Tribolium castaneum of the present application can not only produce β-alanine but also produce pantothenic acid more efficiently.

[0167] Example 3. Construction of a mutant PanD expression vector having aspartate 1-decarboxylase activity

[0168] In order to confirm that a mutation at the position corresponding to position 139 of the PanD protein derived from Tribolium castaneum (wild type, SEQ ID NO: 51) is important for increasing the productivity of β-alanine, as confirmed in Example 1, a mutation in which the phenylalanine at position 139 of the PanD protein derived from Tribolium castaneum (wild type, SEQ ID NO: 51) was substituted with another amino acid was constructed, and the effect was confirmed.

[0169] PCR was performed using the wild-type red castaneum panD gene as a template and using the sequence primers in Table 3, and amplifying the DNA fragment encoding aspartate 1-decarboxylase. PCR was performed using PfuUltraTM high reliability DNA polymerase (Stratagene), and was performed under the conditions of 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute, repeated 30 times. As a result, a DNA fragment of 430bp in the 5' upstream region and a DNA fragment of 1224bp in the 3' downstream region were obtained, focusing on the mutation of the gene encoding aspartate 1-decarboxylase (mutation in the codon encoding the 139th residue of the PanD protein).

[0170] To obtain the PLM1 promoter derived from Corynebacterium glutamicum, PCR was performed in the same manner as above using genomic DNA of Corynebacterium glutamicum (ATCC13032) as a template and using primers of SEQ ID NOs: 39 and 40 to obtain a promoter DNA fragment.

[0171] [Table 3]

[0172] List of primers used for saturation mutagenesis

[0173]

[0174] The pECCG117 (Korean Patent No. 10-0057684) vector and DNA fragments (each panD, PLM1 promoter) treated with restriction endonuclease BamHI and heat-treated at 65°C for 20 minutes were cloned using TaKaRa's Infusion Cloning Kit according to the provided manual so that the molar concentration (M) was 2:1:1:1 (pECCG117 vector: panD upstream region: pan downstream region: promoter) to obtain plasmids, and the names of the 11 plasmids obtained above and the information of the introduced genes are given in Table 4.

[0175] [Table 4]

[0176] List of mutation vectors at position 139

[0177]

[0178]

[0179] Example 4. Evaluation of pantothenate production capacity of mutant PanD

[0180] The 19 mutant plasmids and pECCG117-panD (WT) constructed in Example 3 were introduced into the Corynebacterium glutamicum ATCC13032ΔpanB::panB (EC) strain by the electric pulse method, and then various transformed strains were obtained after coating in a selection medium containing 25 mg / L kanamycin. Thereafter, shake flask evaluation was performed by the same method as in Example 2, and the results are shown in Table 5 below.

[0181] [Table 5]

[0182] Pantothenic acid production ability of strains introduced by saturation mutagenesis

[0183]

[0184] As shown in the above Table 5, all strains into which the wild-type panD was introduced (ATCC 13032ΔpanB::panB(EC)pECCG117-panD(WT)) and strains into which the mutant panD was introduced had increased pantothenate-producing activity.

[0185] In particular, it was confirmed that a total of 10 mutant strains (F139T, F139A, F139S, F139L, F139I, F139V, F139D, F139G, F139H, F139Y) including a mutant strain expressing a mutant PanD (wherein the amino acid at position 139 of the wild-type PanD protein was mutated to tyrosine) produced higher levels of pantothenic acid than the strain expressing the wild-type PanD protein, wherein one of the mutant strains (F139Y) produced more than twice as much pantothenic acid as the wild-type. The results can confirm that position 139 of the PanD protein derived from Tribolium castaneum is an important position for activity, and that when this position is substituted with another amino acid, this position is an important position that affects the production capacity of pantothenic acid.

[0186] In this embodiment, the ATCC 13032ΔpanB::panB(EC)pECCG117-panD(F139Y) strain (named Corynebacterium glutamicum CV03-5004) which was confirmed to have the best pantothenic acid production ability was deposited at the Korea Collection of Microorganisms in Hongje-dong, Seodaemon-gu, Seoul, South Korea on November 23, 2021, and was given the deposit number KCCM13077P.

[0187] According to the above description, it will be understood by those skilled in the art that the present invention can be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the above embodiments should be understood as illustrative and non-restrictive in all aspects. The scope of the present invention should be interpreted as including all changes or modifications from the meaning and scope of the claims described below, and its equivalent concepts are included in the scope of the present invention.

[0188] [Accession number]

[0189] Name of depository: Korea Collection of Microorganisms

[0190] Accession number: KCCM13077P

[0191] Date of deposit: November 23, 2021 (Chinese translation)

[0192] International Form of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0193] To: CJ CheilJedang Co., Ltd.

[0194] 330 Dongho-ro, Jung-gu, Seoul, South Korea

[0195] CJ CheilJedang Center (zip code) 100-400

[0196]

[0197] The above translation does not contradict the original text.

Claims

1. A variant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

2. The polypeptide according to claim 1, wherein The amino acid at residue 139 of the amino acid sequence corresponding to SEQ ID NO:51 is substituted with tryptophan, histidine, tyrosine, alanine, cysteine, proline, serine, leucine, isoleucine, arginine, lysine, valine, methionine, aspartic acid, glutamic acid, glycine, asparagine, glutamine or tyrosine.

3. The polypeptide according to claim 1, wherein The amino acid at residue 139 of the amino acid sequence corresponding to SEQ ID NO: 51 is substituted by threonine, alanine, serine, leucine, isoleucine, valine, aspartic acid, glycine, histidine or tyrosine.

4. The polypeptide according to claim 1, wherein The polypeptide has a sequence identity of greater than 70% and less than 100% with the amino acid sequence of SEQ ID NO:

51. A polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

6. A microorganism comprising a variant polypeptide having aspartate 1-decarboxylase activity or a polynucleotide encoding the variant polypeptide, in which the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

7. The microorganism according to claim 6, wherein The microorganism has an increased ability to produce β-alanine or a β-alanine-derived compound.

8. The microorganism according to claim 6, wherein The microorganism is a microorganism of the genus Corynebacterium sp.

9. The microorganism according to claim 8, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

10. A composition for producing β-alanine or a β-alanine-derived compound, comprising a microorganism, wherein the microorganism comprises a variant polypeptide having aspartate 1-decarboxylase activity or a polynucleotide encoding the variant polypeptide, wherein the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

11. A method for producing β-alanine or a β-alanine-derived compound, comprising culturing a microorganism in a culture medium, wherein the microorganism comprises a variant polypeptide having aspartate 1-decarboxylase activity or a polynucleotide encoding the variant polypeptide, wherein the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO: 51 is substituted by another amino acid.

12. The method for producing β-alanine or a β-alanine derivative compound according to claim 11, further comprising recovering the β-alanine or the β-alanine derivative compound from the cultured microorganism, the culture medium, or both after the culture.

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