Microorganisms having enhanced activity of aspartate 1-decarboxylase derived from Tribolium castaneum and uses thereof

Abstract

The present application provides a microorganism having enhanced activity of aspartate 1-decarboxylase derived from Tribolium castaneum, a composition for producing beta-alanine and / or beta-alanine-derived compounds comprising the microorganism, and a method for producing beta-alanine and / or beta-alanine-derived compounds comprising a step of culturing the microorganism, and has excellent production ability of beta-alanine and / or beta-alanine-derived compounds.

Description

[Technical field]

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0034234 dated March 18, 2022, and all contents disclosed in the documents of the Korean Patent Application are incorporated herein by reference.

[0002] Provided are a microorganism having enhanced activity of aspartate 1-decarboxylase derived from Tribolium castaneum, a composition for producing beta-alanine and / or a beta-alanine derived compound comprising said microorganism, and a method for producing beta-alanine and / or a beta-alanine derived compound comprising a step of culturing said microorganism. [Background technology]

[0003] Beta-alanine is a naturally occurring beta-amino acid with an amino group attached to the beta carbon. Beta-alanine is found naturally in foods such as poultry, meat, and fish, and is used to synthesize carnosine in muscle.

[0004] β-alanine and its derived compounds, such as pantothenic acid, are commercially important substances with applications in diverse industrial fields such as dietary supplements, food, pharmaceuticals, animal feed, etc.

[0005] Therefore, there is a demand for the development of microorganisms having advantageous effects in biotechnologically producing beta-alanine and / or beta-alanine-derived compounds, and techniques for using the same to produce beta-alanine and / or beta-alanine-derived compounds with high efficiency. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent No. 7718205 Summary of the Invention [Problem to be solved by the invention]

[0007] The present application provides a microorganism having enhanced aspartate 1-decarboxylase activity and improved ability to produce beta-alanine and / or beta-alanine-derived compounds.

[0008] The present application provides a composition for producing beta-alanine and / or beta-alanine derived compounds comprising said microorganism.

[0009] The present application provides a method for producing beta-alanine and / or beta-alanine derived compounds comprising culturing said microorganism in a culture medium.

[0010] The present application provides the use of said microorganisms for the production of beta-alanine and / or beta-alanine derived compounds.

[0011] The present application provides the use of said microorganisms for the manufacture of compositions for the production of beta-alanine and / or beta-alanine derived compounds. [Means for solving the problem]

[0012] The present application relates to the use of aspartate 1-decarboxylase, for example aspartate 1-decarboxylase derived from Tribolium castaneum, for the production and / or improvement of the productivity of beta-alanine and / or beta-alanine derived compounds.

[0013] One aspect of the present application provides a microorganism (or strain, recombinant cell) having enhanced activity of aspartate 1-decarboxylase.

[0014] In the present application, aspartate 1-decarboxylase (or PanD protein) may refer to a protein having aspartate 1-decarboxylase activity. The aspartate 1-decarboxylase may be derived from, but is not limited to, red flour beetle (Tribolium castaneum), Escherichia coli, Bacillus subtilis, Serratia rubidaea, Corynebacterium glutamicum, or Pseudomonas sp. In one example, the aspartate 1-decarboxylase may be derived from Tribolium castaneum, and the sequence of the aspartate 1-decarboxylase derived from Tribolium castaneum can be obtained from NCBI, a known database, and can be expressed as, for example, NCBI Reference Sequence: NP_001096055.1. In one example, the aspartate 1-decarboxylase derived from Tribolium castaneum has, contains, or consists of the amino acid sequence of SEQ ID NO: 27, or can essentially consist of the amino acid sequence. Specifically, the aspartate 1-decarboxylase may consist of a polypeptide described as the amino acid sequence of SEQ ID NO: 27.

[0015] In one example, the aspartate 1-decarboxylase protein may have at least 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 with the amino acid sequence set forth in SEQ ID NO: 27, or may include or consist of the sequence. In addition, the aspartate 1-decarboxylase also includes mutants having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and exhibits an activity corresponding to aspartate 1-decarboxylase. For example, the amino acid sequence may have sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally that do not alter the aspartate 1-decarboxylase activity.

[0016] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0017] In the present application, the aspartate 1-decarboxylase may be a polypeptide having aspartate 1-decarboxylase activity encoded by an aspartate 1-decarboxylase gene. The aspartate 1-decarboxylase gene may be derived from, but is not limited to, Red flour beetle (Tribolium castaneum), Escherichia coli, Bacillus subtilis, Serratia rubidaea, Corynebacterium glutamicum, or Pseudomonas sp. In one example, the aspartate 1-decarboxylase gene may be derived from Tribolium castaneum. The sequence of the aspartate 1-decarboxylase gene derived from Tribolium castaneum can be obtained from NCBI, a known database, and can be expressed as, for example, NCBI Reference Sequence: NM_001102585.1. In one example, the aspartate 1-decarboxylase gene derived from Tribolium castaneum has, comprises, consists of, or can essentially consist of the nucleic acid sequence of SEQ ID NO: 38. Specifically, the aspartate 1-decarboxylase gene may consist of the nucleic acid sequence of SEQ ID NO: 38. In one example, the aspartate 1-decarboxylase gene may comprise or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the nucleic acid sequence of SEQ ID NO: 38.

[0018] In this application, a polynucleotide or polypeptide "having, comprising, consisting of, or essentially consisting of a specific nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence (base sequence) or amino acid sequence, and can be interpreted as including a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) have been added to the specific nucleic acid sequence (base sequence) or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or not excluding the mutations). In one example, a polynucleotide or polypeptide "having, comprising, consisting of, or consisting essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide (i) essentially comprises the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of or essentially comprises a nucleic acid sequence or amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology or identity to the specific nucleic acid sequence (base sequence) or amino acid sequence, and maintains its original function and / or a desired function. In one example, the desired function can mean a function of increasing (improving) or imparting the ability of a microorganism to produce beta-alanine and / or a beta-alanine-derived compound.

[0019] As used herein, 'homology' or 'identity' means the degree of similarity between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0020] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to the entire sequence or a portion thereof under moderate or high stringent conditions. It is understood that hybridization also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0021] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, e.g., Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used to determine the sequence, 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 versions) (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 ETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Database Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity, or identity.

[0022] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48:443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program can be defined as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0023] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and may be a microorganism in which a specific mechanism has been enhanced or weakened by, for example, inserting an exogenous gene or enhancing or weakening the activity of an endogenous gene, and may be a microorganism that contains a genetic modification for producing a desired polypeptide, protein, or product (e.g., aspartate 1-decarboxylase).

[0024] In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to its endogenous activity. The term "enhancement" can be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase can all include exhibiting an activity that was not originally possessed, or exhibiting an activity that is improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" means the activity of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before transformation, when the trait is changed by genetic mutation due to natural or artificial factors. This term can be used interchangeably with "activity before modification." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before transformation.

[0025] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression amount) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from an increase in the level of activity, expression amount, or amount of a product resulting from the activity of the polypeptide.

[0026] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the unmodified microorganism. Specifically, the activity of the polypeptide can be enhanced by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to those skilled in the art, but is 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.).

[0027] Specifically, the enhancement of the polypeptide of the present application is 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome that encodes the polypeptide with a sequence with strong activity; 3) modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene to encode a modified polypeptide so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0028] More specifically, The 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the increase may be achieved by introducing one or more copies of a polynucleotide encoding the polypeptide into a chromosome in a host cell. The introduction into a chromosome may be achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0029] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence having a stronger activity may be, for example, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or a replacement with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

[0030] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent US7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US10584338B2), O2 promoter (US Patent US10273491B2), tkt promoter, and yccA promoter.

[0031] The modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, a replacement with a base sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0032] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or a replacement with an improved amino acid sequence or polynucleotide sequence having a stronger activity or an improved amino acid sequence or polynucleotide sequence having an increased activity. The replacement may be specifically, but is not limited to, by inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may additionally include a selection marker for checking whether or not the chromosome is inserted. The selection marker is as described above.

[0033] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell to produce a polypeptide and increase its activity.

[0034] The codon optimization of the polynucleotide encoding the polypeptide (7) may be codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription or translation in a host cell.

[0035] The step 8) of analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, determining candidate template proteins based on the degree of sequence similarity by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of base proteins is stored, confirming the structure based on the sequence similarity, and selecting and modifying exposed sites to be modified or chemically modified.

[0036] Such enhanced polypeptide activity may refer to, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide expressed relative to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0037] In the present application, the term "attenuation" of a polypeptide refers to a concept that includes a decrease in activity or no activity compared to the intrinsic activity. The term "attenuation" can be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0038] The attenuation may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide (or protein, for example, aspartate 1-decarboxylase), where the overall polypeptide activity level and / or concentration (expression amount) in the cell is lower than that of a wild-type strain due to inhibition of gene expression or translation into the polypeptide of the encoding polynucleotide, where the expression of the polynucleotide does not occur at all, and / or where the polypeptide activity is absent even if the polynucleotide is expressed. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, a wild-type or an unmodified microorganism before transformation, when the trait is changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The activity of a polypeptide is "inactivated, deficient, reduced, downregulated, decreased, attenuated" compared to the intrinsic activity, and means that the activity of a specific polypeptide is lowered compared to that of a parent strain or an unmodified microorganism before transformation.

[0039] The activity of such a polypeptide (or protein, e.g., aspartate 1-decarboxylase) can be attenuated by any method known in the art, including, but not limited to, by application of a variety of methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0040] Specifically, the weakening of a polypeptide (or protein, such as aspartate 1-decarboxylase; hereinafter, referred to as a polypeptide) 1) Deletion of all or part of a gene encoding a polypeptide; 2) modifying an expression control region (or expression control sequence) so that expression of a gene encoding a polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence); 4) modification of a gene sequence encoding a polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid base sequence of the polypeptide gene to encode a modified polypeptide such that the activity of the polypeptide is eliminated or attenuated); 5) modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach; 8) Reverse transcription engineering (RTE) of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0041] For example, the 1) deletion of a part or the whole of the gene encoding a polypeptide may be removal of the entire polynucleotide encoding a target polypeptide endogenous to a chromosome, replacement with a polynucleotide having a partial deletion of nucleotides, or replacement with a marker gene.

[0042] In addition, the modification of the expression regulatory region (or expression regulatory sequence) may be a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or a replacement with a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0043] In addition, the modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a replacement with a base sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0044] In addition, the modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to weaken the activity of the polypeptide, or a replacement with an improved amino acid sequence or polynucleotide sequence having a weaker activity or an improved amino acid sequence or polynucleotide sequence having no activity. For example, but not limited to, gene expression can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon. The "stop codon" is a codon on mRNA that does not specify an amino acid and acts as a signal to inform the end of the protein synthesis process, and generally, UAA, UAG, and UGA can be used as stop codons.

[0045] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide 6) can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol.1(1)1986].

[0046] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach may disable or slow down mRNA translation.

[0047] 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) can create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0048] The microorganism of the present application may be, but is not limited to, a microorganism genetically modified (e.g., a recombinant microorganism) through a vector so that the activity of aspartate 1-decarboxylase or a polynucleotide encoding the same is enhanced. The vector is as described above.

[0049] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having the ability to produce beta-alanine and / or beta-alanine derived compounds or having an improved ability (or production amount) to produce beta-alanine and / or beta-alanine derived compounds.

[0050] The microorganism of the present application may be, but is not limited to, a microorganism that does not naturally have the ability to produce beta-alanine and / or beta-alanine-derived compounds, or a microorganism that has the ability to produce beta-alanine and / or beta-alanine-derived compounds and has been imparted or improved with the ability to produce beta-alanine and / or beta-alanine-derived compounds by introducing or enhancing the activity of aspartate 1-decarboxylase into the microorganism. The microorganism imparted or improved with the ability to produce beta-alanine and / or beta-alanine-derived compounds may be a microorganism into which aspartate 1-decarboxylase derived from Tribolium castaneum has been introduced.

[0051] The microorganism (or strain, recombinant cell) having improved ability to produce beta-alanine or a beta-alanine-derived compound (or production amount) or having the ability to produce beta-alanine or a beta-alanine-derived compound may mean that the microorganism (or strain, recombinant cell) has improved ability to produce beta-alanine or a beta-alanine-derived compound compared to an unmodified microorganism, a cell before recombination, a parent strain, a wild-type strain, and / or a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced, or that the ability to produce beta-alanine or a beta-alanine-derived compound has been imparted, unlike an unmodified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain that does not have the ability to produce beta-alanine or a beta-alanine-derived compound.

[0052] According to an embodiment, a microorganism having enhanced aspartate 1-decarboxylase activity may have improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds compared to a microorganism before enhancement, i.e., an unmodified microorganism of the same species. In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, but may refer to a wild-type strain or a natural strain itself, or a strain before its phenotype is changed by genetic mutation due to natural or artificial factors. For example, according to an embodiment, the unmodified microorganism may refer to a strain in which the activity of aspartate 1-decarboxylase is not enhanced or before it is enhanced (or a strain in which a mutation that induces enhancement of the activity of aspartate 1-decarboxylase is not introduced or before it is introduced). The term "unmodified microorganism" may be used interchangeably with "unmodified strain", "unmodified microorganism", "non-mutated strain", "unmodified strain", "non-mutated microorganism" or "reference microorganism". The fact that the activity of aspartate 1-decarboxylase is enhanced is as described above. In one example, the unmodified microorganism, which is a subject strain for comparing the increase in the ability to produce beta-alanine and / or beta-alanine-derived compounds, may be a wild-type Corynebacterium glutamicum ATCC13032 strain.

[0053] According to one example, the microorganism having enhanced aspartate 1-decarboxylase activity may be a microorganism into which aspartate 1-decarboxylase derived from Tribolium castaneum has been introduced, and the microorganism may have a further improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds compared to a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced. The aspartate 1-decarboxylase derived from another microorganism may be derived from Escherichia coli, Bacillus subtilis, Serratia rubidaea, Corynebacterium glutamicum, or Pseudomonas sp.

[0054] The microorganism (or strain, recombinant cell) may further include a mutation that increases the production of beta-alanine and / or beta-alanine derived compounds, and the position of the mutation and / or the type of gene and / or protein to be mutated may include any one that increases the production of beta-alanine and / or beta-alanine derived compounds without limitation. The recombinant cell may be used without limitation as long as it is a cell that can be transformed.

[0055] The beta-alanine derived compound may be one or more selected from the group consisting of β-nitropropanoate, β-amino-propionitrile, N-acetyl-β-alanine, L-aspartate, anserine, spermine, carnosine, β-alanyl arginine, quinolinate, pantothenate (or pantothenic acid), malonate semialdyhyde, malonate, acetylene-monocarboxylate, and the like, but is not limited thereto.

[0056] As an example, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) has an ability to produce beta-alanine and / or beta-alanine-derived compounds of about 10% or more, about 20% or more, about 30% or more, about 50% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, about 100% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 5 ... % or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, about 1,000% or more, about 1,500% or more, about 2,000% or more, about 2,500% or more, or about 3,000% or more, and in one example, may be an increase of about 33.3% or more, about 100% or more, about 175% or more, about 243.75% or more, about 266.6% or more, about 300% or more, about 450% or more, about 500% or more, about 587.5% or more, about 700% or more, or about 2,650% or more, but is not limited thereto.

[0057] In another example, the microorganism (or strain, recombinant cell) having increased production ability (or production amount) has an increased production ability (or production amount) of beta-alanine and / or beta-alanine-derived compounds of about 1.1 times or more, about 1.3 times or more, about 1.5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, compared to the parent strain before mutation, the unmodified microorganism, or a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced. The increase may be about 9-fold or more, about 10-fold or more, about 15-fold or more, about 20-fold or more, about 25-fold or more, or about 30-fold or more (the upper limit is not particularly limited, and may be, for example, about 1,000-fold or less), and in one example, the increase may be about 1.3-fold or more, about 2-fold or more, about 2.75-fold or more, about 3.43-fold or more, about 3.6-fold or more, about 4-fold or more, about 5.5-fold or more, about 6-fold or more, about 6.8-fold or more, about 8-fold or more, or about 27.5-fold or more, but is not limited thereto.

[0058] In another example, the microorganism (or strain, recombinant cell) having increased production ability (or production amount) has a beta-alanine and / or beta-alanine-derived compound production ability (or production amount) of about 0.5 g / L or more, about 1 g / L or more, about 1.5 g / L or more, about 2.0 g / L or more, about 2.5 g / L or more, about 3 g / L or more, about 3.5 g / L or more, about 4 g / L or more, about 4.5 g / L or more, about 5 g / L or more, about 5.5 g / L or more, about 6 g / L or more, compared to a pre-mutation parent strain, an unmodified microorganism, or a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced. or more, about 7 g / L or more, about 8 g / L or more, about 9 g / L or more, about 10 g / L or more, about 15 g / L or more, about 20 g / L or more, about 25 g / L or more, about 30 g / L or more (there is no particular limit to the upper limit, and it may be, for example, about 100 g / L or less), and in one example, it may be an increase of about 0.6 g / L or more, about 1.2 g / L or more, about 1.8 g / L or more, about 2 g / L or more, about 2.4 g / L or more, about 3.5 g / L or more, about 3.9 g / L or more, about 4 g / L or more, about 4.5 g / L or more, about 4.7 g / L or more, or about 5.3 g / L or more, but is not limited thereto.

[0059] More specifically, the microorganism (or strain, recombinant cell) having the increased production ability (or production amount) has an increased production ability (or production amount) of about 33.3%, about 100%, about 175%, about 243.75%, about 266.6%, about 300%, about 450%, about 500%, about 587%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1200%, about 1500%, about 1600%, about 1750%, about 243.75%, about 266.6%, about 300%, about 450%, about 500%, about 1800%, about 1900%, about 2000%, about 2100%, about 2200%, about 2300%, about 2400%, about 2500%, about 2600%, about 2700%, about 2800%, about 2900%, about 3000%, about 3000%, about 3100%, about 3200%, about 3300%, about 3400%, about 3500%, about 3600%, about 3700%, about 3800%, about 3900%, about 4000%, about 4100%, about 4200%, about 4300%, about 4400%, about 4500%, about 4600%, about 4700%, about 4800%, about 4900%, about 50 ... The increase may be, but is not limited to, about 0.5%, about 700%, or about 2,650% (or about 1.3-fold, about 2-fold, about 2.75-fold, about 3.43-fold, about 3.6-fold, about 4-fold, about 5.5-fold, about 6-fold, about 6.8-fold, about 8-fold, or about 27.5-fold; or about 0.6 g / L, about 1.2 g / L, about 1.8 g / L, about 2 g / L, about 2.4 g / L, about 3.5 g / L, about 3.9 g / L, about 4 g / L, about 4.5 g / L, about 4.7 g / L, about 5.3 g / L).

[0060] The term "about" includes, but is not limited to, all numerical values ​​in a range equal to or similar to the numerical value preceding the term about, including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc.

[0061] In one example, the microorganism having enhanced aspartate 1-decarboxylase activity may be a Corynebacterium sp. microorganism. The microorganism of the genus Corynebacterium is, for example, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and / or Corynebacterium flavescens.

[0062] Yet another aspect of the present application provides a method for producing (or a method for manufacturing) beta-alanine and / or beta-alanine derived compounds, comprising culturing a microorganism of the present application in a medium.

[0063] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application can include culturing the microorganism of the present application in a medium. The microorganism, beta-alanine derived compounds, etc. of the present application are as described above.

[0064] In the present application, "culturing" refers to growing a microorganism having enhanced aspartate 1-decarboxylase activity, such as a Corynebacterium glutamicum strain, under appropriately controlled environmental conditions. The culturing process of the present application can be performed using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch type.

[0065] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing a microorganism having enhanced aspartate 1-decarboxylase activity of the present application, for example, a Corynebacterium glutamicum strain, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular limitation, and the microorganism of the present application can be cultured under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while controlling temperature, pH, etc.

[0066] Specifically, the culture medium for the microorganism of the present application, for example, a Corynebacterium sp. strain, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0067] In the present application, the carbon source may 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, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residue, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other suitable amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0068] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0069] The phosphorus source may include potassium monophosphate, potassium diphosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may also include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0070] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foam generation may be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain anaerobic and microaerobic states, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0071] In the culture of the present application, the culture temperature can be maintained at 20 to 45° C., specifically 25 to 40° C., and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0072] The beta-alanine and / or beta-alanine derived compounds produced by the cultures of the present application may be secreted into the medium or remain intracellularly.

[0073] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application may additionally include, for example prior to the culturing step, a step of preparing a microorganism (strain) of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order).

[0074] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application may further include a step of recovering beta-alanine and / or beta-alanine derived compounds from the culture medium (culture medium) or microorganism (e.g., Corynebacterium sp. strain). The recovery step may be additionally included after the culture step.

[0075] Said recovery may be to collect the desired beta-alanine and / or beta-alanine derived compounds using a suitable method known in the art, such as a batch, continuous or fed-batch culture method of the microorganism of the present application. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination of these methods can be used, and the desired beta-alanine and / or beta-alanine derived compounds can be collected from the medium or the microorganism using a suitable method known in the art.

[0076] In addition, the method for producing beta-alanine and / or beta-alanine derived compounds of the present application may additionally include a purification step. The purification may be carried out using any suitable method known in the art. In one example, when the method for producing beta-alanine and / or beta-alanine derived compounds of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously in any order, or may be carried out simultaneously or integrated into one step, but is not limited thereto.

[0077] Another aspect of the present application provides a composition for producing beta-alanine and / or beta-alanine derived compounds, comprising a microorganism of the present application, a culture medium in which the microorganism is cultured, or a combination thereof.

[0078] The compositions of the present application may additionally contain any suitable excipients commonly used in compositions for the production of beta-alanine and / or beta-alanine derived compounds, such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0079] In the composition of the present application, the microorganism (strain), the medium, the beta-alanine derived compound, etc. are as described in other embodiments above.

[0080] Another aspect of the present application provides a use of the present microorganism for the production of beta-alanine and / or beta-alanine derived compounds or for the manufacture of a composition for the production of beta-alanine and / or beta-alanine derived compounds, wherein the present microorganism, the beta-alanine and / or beta-alanine derived compounds or the composition for the production of beta-alanine and / or beta-alanine derived compounds are as described above. Effect of the Invention

[0081] The present application provides a microorganism having enhanced activity of aspartate 1-decarboxylase derived from Tribolium castaneum, and the microorganism has excellent ability to produce beta-alanine and / or beta-alanine-derived compounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The present invention will be described in more detail below with reference to the following examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0083] Example 1. Searching and screening for aspartate 1-decarboxylase genes Microbial aspartate 1-decarboxylase (PanD) is classified into two types, that is, a form that is expressed and active, and a form that is activated by the PanD regulatory factor (PanM). Therefore, we performed a gene search for aspartate 1-decarboxylase derived from a microorganism that requires the PanM regulatory factor and a gene search for a microorganism that has an active aspartate 1-decarboxylase.

[0084] Candidate genes and microorganisms that possess aspartate 1-decarboxylase were selected based on organisms that possess aspartate 1-decarboxylase in prokaryotic and eukaryotic cells. Among them, aspartate 1-decarboxylases derived from microorganisms with a biosafety level of 1 were selected as shown in Table 1.

[0085] [Table 1]

[0086] Example 2. Preparation of Corynebacterium sp. microorganisms transfected with aspartate 1-decarboxylase derived from a foreign microorganism After extracting the genome of the obtained microorganism, a DNA fragment encoding aspartate 1-decarboxylase was PCR-generated using the primer sequences (SEQ ID NOs: 1 and 2, 5 and 6, 7 and 8, 11 and 12, 13 and 14, 15 and 16) in Table 1 as a template. PCR was performed using PfuUltraTM high-fidelity DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 3 minutes, repeated 30 times. As a result, aspartate 1-decarboxylase derived from each microorganism was obtained. In addition, a DNA fragment encoding the PanD regulator (PanM) was PCR-generated using the primer sequences (SEQ ID NOs: 3 and 4, 9 and 10) in Table 1 as a template using the genomes of Escherichia coli and Serratia rubidaea microorganisms. PCR was performed using PfuUltraTM high-fidelity DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95℃ for 30 seconds; annealing at 55℃ for 30 seconds; and polymerization at 72℃ for 3 minutes, repeated 30 times. As a result, PanD regulators from E. coli and Serratia rubidaea were obtained. To obtain the lysC promoter from Corynebacterium glutamicum, the promoter was PCRed in the same manner as above using primers 17 and 18 with the genomic DNA of Corynebacterium glutamicum as a template to obtain a DNA fragment. The DNA fragment was treated with the restriction enzyme BamHI and then heat-treated at 65℃ for 20 minutes, and the pECCG117 (Korea Patent No. 10-0057684) vector and the DNA fragment were cloned according to the manual provided by the Infusion Cloning Kit of Takara to obtain a plasmid, and the vector name and the information of the introduced gene are shown in Table 1.

[0087] The six vectors thus constructed were transformed into Corynebacterium glutamicum ATCC13032 by electroporation to generate strains expressing exogenous aspartate 1-decarboxylase (PanD).

[0088] Example 3. Beta-alanine production ability of Corynebacterium sp. microorganisms expressing foreign microbial aspartate 1-decarboxylase The strain obtained in Example 2 was inoculated together with the parent strain into a 250 ml corner baffle flask containing 25 ml of a production medium having the following composition, and cultured at 33° C. for 48 hours with shaking at 200 rpm. The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and the supernatant was diluted 1 / 10 with TDW (triple distilled water), and then analyzed by HPLC to measure the concentration of beta-alanine. The results are shown in Table 2 below.

[0089] <Production medium> Glucose 10%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 10 mg / l, manganese sulfate monohydrate 6.7 mg / l, biotin 50 μg / l, thiamine·HCl 100 μg / l, pH 7.2

[0090] [Table 2]

[0091] As a result, as shown in Table 2, it was confirmed that the parent strain Corynebacterium glutamicum ATCC13032 did not produce beta-alanine, while the strain ATCC13032 pECCG117-panD(CG) expressing aspartate 1-decarboxylase from Corynebacterium glutamicum produced about 1.2 g / L of beta-alanine. Among the strains expressing aspartate 1-decarboxylase derived from foreign microorganisms, the strain ATCC13032 pECCG117-panD(TC) expressing aspartate 1-decarboxylase derived from Tribolium castaneum showed the highest beta-alanine productivity at 2.4 g / L.

[0092] The above results indicate that, among the six enzymes selected in the present invention, aspartate 1-decarboxylase derived from Tribolium castaneum can produce beta-alanine more efficiently.

[0093] The ATCC13032 pECCG117-panD(TC) strain (named Corynebacterium glutamicum CV03-5003), which was confirmed in this embodiment to have the best beta-alanine production ability, was deposited at the Korea Microorganism Collection, Hongje-dong, Seodaemun-gu, Seoul, Republic of Korea on November 23, 2021, and was assigned the deposit number KCCM13076P.

[0094] Example 4. Investigation of the ability of Corynebacterium spp. microorganisms expressing aspartate 1-decarboxylase derived from foreign microorganisms to produce beta-alanine-derived compounds In order to confirm the ability of aspartate 1-decarboxylase from Tribolium castaneum to produce a beta-alanine-derived compound (pantothenic acid) as confirmed in Example 3, a microorganism with enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase (or PanB protein) was created.

[0095] First, a vector was constructed to delete the panB gene present in the parent strain. PCR was performed using primers SEQ ID NO:28 and 29 and SEQ ID NO:30 and 31 with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. PCR was performed under the following conditions: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 30 times. As a result, gene fragments of 1000 bp upstream of the panB gene and 1000 bp downstream of the panB gene were obtained, and each amplified product was purified using QIAGEN's PCR Purification kit and used as the insert DNA fragment for vector construction.

[0096] The pDCM2 (Korea Patent No. 2278000) vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the DNA fragments (a 1000 bp gene fragment from the upstream region of the panB gene and a 1000 bp gene fragment from the downstream region of the panB gene) were cloned at a molar concentration (M) of 2:1:1 using the Infusion Cloning Kit from Takara according to the manual provided, to produce the vector pDCM2_ΔpanB for deleting the panB gene on the chromosome.

[0097] To prepare the panB gene from E. coli, PCR was performed using primers 32 and 33 with the genomic DNA of E. coli K12 wild-type strain (KCTC1116) as a template. PCR was performed by repeating denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute 30 times, resulting in a 795 bp DNA fragment. To obtain the lysC promoter from Corynebacterium glutamicum, PCR was performed in the same manner as in the previous example with primers 34 and 35 with the genomic DNA of Corynebacterium glutamicum as a template to obtain a DNA fragment. The pDCM2_ΔpanB vector was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The DNA fragment obtained was then cloned using the Infusion Cloning Kit from Takara Corporation according to the instructions provided, at a molar concentration (M) of 2:1:1, to produce the vector pDCM2_ΔpanB::panB(EC) for introducing the panB gene derived from E. coli into the chromosome.

[0098] The constructed vector pDCM2_ΔpanB::panB(EC) was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain (ΔpanB::panB(EC)) in which E. coli-derived panB was introduced into the chromosome was obtained through a second crossover process. The appropriate substitution of E. coli-derived panB was confirmed using the MASA (Mutant Allele Specific Amplification) PCR technique (Takeda et al., Hum. Mutation, 2, 112-117(1993)) with the following primer combinations. That is, the strains that were amplified with the primer combinations (sequences 36 and 35 and sequences 37 and 32) corresponding to E. coli panB were primarily selected, and the panB sequences of the selected strains were secondarily confirmed by analyzing the primer combinations of sequence numbers 36 and 37.

[0099] The plasmid obtained in Example 1 was introduced into the ATCC13032 ΔpanB::panB(EC) strain, and the parent strain and the above strain were inoculated into a 250 ml corner baffle flask containing 25 ml of a production medium prepared according to the composition of the above Example, and then cultured at 33° C. for 48 hours with shaking at 200 rpm to measure pantothenic acid productivity.

[0100] [Table 3]

[0101] As a result, as shown in Table 3, the parent strain, Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC), hardly produced any pantothenic acid, while the strain expressing aspartate 1-decarboxylase from Corynebacterium glutamicum (ATCC13032 ΔpanB::panB(EC) pECCG117-panD(CG)) produced approximately 2.0 g / L of pantothenic acid. Among the strains expressing aspartate 1-decarboxylase derived from foreign microorganisms, ATCC13032 ΔpanB::panB(EC) pECCG117-panD(TC), which expressed aspartate 1-decarboxylase derived from Tribolium castaneum, showed the highest pantothenic acid productivity at 5.5 g / L.

[0102] The above results indicate that, among the six enzymes selected in the present invention, aspartate 1-decarboxylase derived from Tribolium castaneum can produce not only beta-alanine but also pantothenic acid more efficiently.

[0103] From the above description, it should be understood by those skilled in the art to which the present invention pertains that the present invention can be embodied in other specific forms without changing the technical idea or essential features thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below and their equivalent concepts rather than the above detailed description. [Accession number]

[0104] Name of depository institution: Korea Microorganism Collection Center Accession number: KCCM13076P Date of acceptance: 20211123 TIFF2025509856000004.tif206168

Claims

1. A microorganism of the genus Corynebacterium into which aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the same has been introduced, which produces beta-alanine or a beta-alanine-derived compound.

2. The microorganism according to claim 1 , wherein the aspartate 1-decarboxylase is a PanD protein.

3. The microorganism according to claim 1, wherein the aspartate 1-decarboxylase comprises an amino acid sequence represented by SEQ ID NO:27 or an amino acid sequence having 95% or more sequence identity thereto.

4. The microorganism of claim 1, wherein the aspartate 1-decarboxylase is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:

38.

5. The microorganism according to any one of claims 1 to 4, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

6. The microorganism according to any one of claims 1 to 4, wherein the microorganism has an increased ability to produce beta-alanine or a beta-alanine-derived compound compared to a parent strain or a wild-type strain in which the aspartate 1-decarboxylase or a polynucleotide encoding the same has not been introduced.

7. A method for producing beta-alanine or a beta-alanine-derived compound, comprising the step of culturing in a medium a microorganism of the genus Corynebacterium into which aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the same has been introduced.

8. 8. The method of claim 7, further comprising recovering the beta-alanine or beta-alanine derived compound from the culture medium or microorganism.

9. A composition for producing beta-alanine or a beta-alanine-derived compound, comprising a Corynebacterium microorganism into which aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the same has been introduced.

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