Microorganisms with enhanced aspartate 1-decarboxylase activity derived from confused flour beetle, and their applications

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] Mutual citation with related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0034234 filed on March 18, 2022, and all the contents disclosed in the literature of the Korean Patent Application are incorporated herein by reference.

[0002] Provided are a microorganism having enhanced activity of aspartate 1-decarboxylase derived from Cocos nucifera var. typica, a composition for producing beta-alanine and / or a beta-alanine-derived compound containing the microorganism, and a method for producing beta-alanine and / or a beta-alanine-derived compound including culturing the microorganism.

Background Art

[0003] Beta-alanine is a naturally produced beta-amino acid in which an amino group is bonded to the beta-carbon. Beta-alanine is naturally found in foods such as poultry, meat, and fish and is used to synthesize carnosine in muscles.

[0004] Beta-alanine and beta-alanine-derived compounds such as pantothenic acid are one of the commercially important substances applied in various industrial fields such as health supplements, foods, pharmaceuticals, animal feeds, and the like.

[0005] Therefore, there is a need to develop a microorganism having an advantageous effect for biotechnologically producing beta-alanine and / or a beta-alanine-derived compound and a technique for highly efficiently producing beta-alanine and / or a beta-alanine-derived compound using the same.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] This application provides a microorganism in which the activity of aspartate 1-decarboxylase is enhanced and the ability to produce beta-alanine and / or beta-alanine-derived compounds is improved.

[0008] This application provides a composition for producing beta-alanine and / or beta-alanine-derived compounds, comprising the aforementioned microorganism.

[0009] This application provides a method for producing beta-alanine and / or beta-alanine-derived compounds, comprising the step of culturing the microorganism in a culture medium.

[0010] This application provides applications for use in the production of beta-alanine and / or beta-alanine-derived compounds by the aforementioned microorganisms.

[0011] This application provides applications for use in the production of compositions for the production of beta-alanine and / or beta-alanine-derived compounds from the aforementioned microorganisms. [Means for solving the problem]

[0012] This application relates to the use of aspartate 1-decarboxylase, for example, aspartate 1-decarboxylase derived from the confused flour beetle (Tribolium castaneum), for the production and / or improvement of the production capacity of beta-alanine and / or beta-alanine-derived compounds.

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

[0014] In this application, aspartate 1-decarboxylase (or PanD protein) can mean 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. For example, the aspartate 1-decarboxylase may be derived from the confused flour beetle, and the sequence of the confused flour beetle-derived aspartate 1-decarboxylase can be obtained from the NCBI, a known database, and can be expressed as, for example, NCBI Reference Sequence: NP_001096055.1. For example, the confused flour beetle-derived aspartate 1-decarboxylase may have, contain, consist of, or be essentially derived from the amino acid sequence of SEQ ID NO: 27. Specifically, the aspartate 1-decarboxylase may consist of a polypeptide described as having the amino acid sequence of SEQ ID NO: 27.

[0015] For example, the aspartate 1-decarboxylase protein may have, but is not limited to, a homology or identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 98.9%, 99%, 99.1%, 99.3%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence described in SEQ ID NO: 27, or it may contain or consist of the sequence. Furthermore, any mutant amino acid sequence having such homology or identity and exhibiting efficacy corresponding to aspartate 1-decarboxylase is also included in the definition of aspartate 1-decarboxylase, including those with partially deleted, modified, substituted, conserved substituted, or added amino acid sequences. For example, the amino acid sequence may have additions or deletions of sequences that do not alter aspertate 1-decarboxylase activity, spontaneously occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within it.

[0016] The aforementioned “conservative substitution” means replacing one amino acid with another amino acid that has similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0017] In this application, the aspartate 1-decarboxylase may be a polypeptide having aspartate 1-decarboxylase activity encoded by the aspartate 1-decarboxylase gene. The aspartate 1-decarboxylase gene may, but is not limited to, those derived from red flour beetle (Tribolium castaneum), Escherichia coli, Bacillus subtilis, Serratia rubidaea, Corynebacterium glutamicum, or Pseudomonas sp. For example, the aspartate 1-decarboxylase gene may be derived from red flour beetle. The aspartate 1-decarboxylase gene derived from the confused flour beetle can be obtained from the NCBI, a known database, and can be expressed as, for example, NCBI Reference Sequence:NM_001102585.1. For example, the aspartate 1-decarboxylase gene derived from the confused flour beetle may have, contain, consist of, or be essentially composed of the nucleic acid sequence of Sequence ID No. 38. Specifically, the aspartate 1-decarboxylase gene may consist of the nucleic acid sequence of Sequence ID No. 38. For example, the aspartate 1-decarboxylase gene may contain 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 with the nucleic acid sequence of Sequence ID No. 38.

[0018] In this application, the phrase "having, including, consisting of, or essentially consisting of a specific nucleic acid sequence (base sequence) or amino acid sequence" of a polynucleotide or polypeptide can be interpreted as meaning that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and includes (or does not exclude) a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) have been made 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. For example, the statement that a polynucleotide or polypeptide "has, contains, consists of, or is essential to a specific nucleic acid sequence (base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of or essentially contains 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 with the specific nucleic acid sequence (base sequence) or amino acid sequence, and maintains its original function and / or intended function. For example, the intended function can mean the function of increasing (improving) or conferring the ability of microorganisms to produce beta-alanine and / or beta-alanine-derived compounds.

[0019] In this application, '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 interchangeable.

[0020] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, along with a default gap penalty established by the program used. Substantially, homologous or identical sequences can generally hybridize with whole or partial sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing general codons or codons considering codon degeneracy in the polynucleotide.

[0021] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using a known computer algorithm such as the “FASTA” program with default parameters, for example, as in 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), as performed in the EMBOSS package's Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), can be used to determine the results (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, (Including 1994 and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0022] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as those published, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or in Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (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 penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0023] In this application, the term “microorganism (or strain)” includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may include microorganisms in which a particular mechanism has been enhanced or weakened due to causes such as the insertion of an external gene or the enhancement or weakening of the activity of an endogenous gene, and which may include genetic modification for the production of a target polypeptide, protein, or product (e.g., aspartate 1-decarboxylase).

[0024] In this application, the term "enhancement" of polypeptide activity means that the activity of the polypeptide increases compared to its intrinsic activity. The said enhancement can be used interchangeably with terms such as "activation", "up-regulation", "overexpression", "increase", etc. Here, activation, enhancement, up-regulation, overexpression, and increase can all include the fact that the polypeptide comes to exhibit an activity that it did not originally have, or an activity that is improved compared to the intrinsic activity or the activity before modification. The said "intrinsic activity" means the activity of a specific polypeptide that the parental strain or unmodified microorganism had before the trait change when the trait changes due to genetic mutations caused by natural or artificial factors. This can be used interchangeably with "activity before modification". That the activity of a polypeptide "enhances", "up-regulates", "overexpresses", or "increases" compared to its intrinsic activity means that it is improved compared to the activity and / or concentration (expression level) of the specific polypeptide that the parental strain or unmodified microorganism had before the trait change.

[0025] The said enhancement can be achieved by introducing an exogenous polypeptide or through enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the said polypeptide is enhanced can be confirmed from the degree of activity of the polypeptide, the expression level, or the increase in the amount of the product due to the polypeptide activity.

[0026] The enhancement of the activity of the polypeptide can be achieved by applying various methods well-known in the art, and is not limited as long as the activity of the target polypeptide can be strengthened compared to the microorganism before modification. Specifically, it may be, but is not limited to, the use of genetic engineering and / or protein engineering well-known to ordinary technicians in the industry, which are routine methods in molecular biology (for example, 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 1) Increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) Replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having strong activity; 3) Modifying the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide; 4) Modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) Modifying the polynucleotide sequence encoding the polypeptide so that the polypeptide activity is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is enhanced); 6) Introducing a foreign polypeptide showing the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) Codon optimization of the polynucleotide encoding the polypeptide; 8) Analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemically modifying it; 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 increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosome may be, but is not limited to, carried out by introducing into the host cell a vector that can insert the polynucleotide into the chromosomes within the host cell. The vector is as described above.

[0029] The replacement of a gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the regulatory region, thereby causing sequence mutations, or replacement with a sequence having even stronger activity. The regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter.

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

[0031] The nucleotide sequence modification encoding the start codon or 5'-UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon.

[0032] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, thereby causing sequence mutations, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity. The replacement may be, but is not limited to, insertion of a polynucleotide into the chromosome by homologous recombination. The vector used in this case may additionally include a selection marker to confirm the presence or absence of a chromosomal insertion. The selection marker is as described above.

[0033] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence as long as it exhibits the same or similar activity as the polypeptide. A person skilled in the art can appropriately select a known transformation method for the introduction, and the introduction of the polynucleotide into the host cell will generate the polypeptide and increase its activity.

[0034] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of an endogenous polynucleotide so that transcription or translation is increased in the host cell, or optimization of the codon of an exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0035] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying exposed sites or chemically modifying it may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of the base protein is stored to determine a candidate template protein based on the degree of sequence similarity, confirming the structure based on this, and selecting and deforming or modifying exposed sites to be modified or chemically modified.

[0036] Such enhancement of polypeptide activity may, but is not limited to, an increase in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of product produced from said polypeptide.

[0037] In this application, the term “weakening” of a polypeptide encompasses all concepts of reduced or absent activity compared to its endogenous activity. Such weakening can be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0038] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide (or protein, e.g., aspartate 1-decarboxylase), cases where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where the polynucleotide is expressed but the polypeptide is not active. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain before the trait change, the wild type, or the unmodified microorganism when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “pre-modification activity.” “Inactivation, deficiency, reduction, downregulation, decrease, attenuation” of polypeptide activity compared to intrinsic activity means that it has become lower than the activity of a specific polypeptide originally possessed by the parent strain before the trait change or the unmodified microorganism.

[0039] The weakening of the activity of such polypeptides (or proteins, e.g., aspertate 1-decarboxylase) can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (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 polypeptides (or proteins, such as aspertate 1-decarboxylase; hereinafter referred to as polypeptides) 1) Deletion of all or part of the gene encoding a polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide; 3) Modification of the amino acid sequence constituting the polypeptide so as to remove or weaken the polypeptide's activity (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence); 4) Modification of the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid sequence of the polypeptide gene so as to encode a polypeptide modified so that the polypeptide activity is removed or weakened); 5) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily 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 in a polypeptide-encoding gene to form a secondary structure that cannot be attached to a ribosome; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (reverse transcription engineering, RTE); or 9) Two or more combinations selected from 1) to 8) above are also acceptable, but are not particularly limited to these.

[0041] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0042] Furthermore, the modification of the regulatory region (or regulatory sequence) described in 2) above may involve mutagenesis on the regulatory region (or regulatory sequence) by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having weaker activity. The regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0043] Furthermore, the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may, for example, be replaced with a base sequence encoding another start codon with an even lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0044] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence modified to have even weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity. For example, gene expression can be inhibited or weakened by introducing intracellular mutations in a polynucleotide sequence to form a stop codon, but are not limited to this. The “stop codon” is a codon on mRNA that does not specify an amino acid and acts as a signal that the protein synthesis process has ended, and generally three types of codons can be used as stop codons: UAA, UAG, and UGA.

[0045] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide, see, 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) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure to which ribosome attachment is impossible may make mRNA translation impossible or reduce its rate.

[0047] The addition of a promoter that is 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) may weaken the activity of the transcription of the polypeptide gene by creating complementary antisense nucleotides.

[0048] The microorganisms of this application may, but are not limited to, microorganisms genetically modified through a vector to enhance the activity of aspartate 1-decarboxylase or the polynucleotide encoding it (e.g., recombinant microorganisms). The vectors are as described above.

[0049] The microorganism (or strain, recombinant cell) of this application may be a microorganism that has the ability to produce beta-alanine and / or beta-alanine-derived compounds, or a microorganism in which the ability (or production volume) of beta-alanine and / or beta-alanine-derived compounds has been improved.

[0050] The microorganisms of this application may be, but are not limited to, microorganisms that do not naturally possess the ability to produce beta-alanine and / or beta-alanine-derived compounds, or microorganisms that possess the ability to produce beta-alanine and / or beta-alanine-derived compounds, in which the activity of aspartate 1-decarboxylase has been introduced or enhanced to confer or improve the ability to produce beta-alanine and / or beta-alanine-derived compounds. The microorganisms in which the ability to produce beta-alanine and / or beta-alanine-derived compounds has been conferred or improved may be microorganisms into which aspartate 1-decarboxylase derived from confused flour beetle has been introduced.

[0051] The fact that the microorganism (or strain, recombinant cell) has improved ability (or production volume) to produce beta-alanine or beta-alanine-derived compounds, or has the ability to produce beta-alanine or beta-alanine-derived compounds, can mean that the microorganism (or strain, recombinant cell) has improved ability to produce beta-alanine or beta-alanine-derived compounds compared to an unmodified microorganism, a pre-recombination cell, a parent strain, a wild-type strain, and / or a microorganism into which aspartate 1-decarboxylase from another microorganism has been introduced, or that it has been conferred the ability to produce beta-alanine or beta-alanine-derived compounds, unlike an unmodified microorganism, a pre-recombination cell, a parent strain, and / or a wild-type strain that does not have the ability to produce beta-alanine or beta-alanine-derived compounds.

[0052] A microorganism in which aspartate 1-decarboxylase activity is enhanced, for example, may have improved (increased) beta-alanine and / or beta-alanine-derived compound production capacity compared to the pre-enhancement microorganism, i.e., the same unmodified microorganism. In this application, “unmodified microorganism” may mean the wild-type strain or the native strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors, rather than excluding strains containing mutations that can occur naturally in microorganisms. For example, the unmodified microorganism may mean, for example, a strain in which aspartate 1-decarboxylase activity is not enhanced or before it is enhanced (or a strain in which a mutation that induces enhancement of aspartate 1-decarboxylase activity is not introduced or before it is introduced). The “unmodified microorganism” can be used interchangeably with “pre-modification strain,” “pre-modification microorganism,” “non-mutant strain,” “unmodified strain,” “non-mutant microorganism,” or “reference microorganism.” The enhancement of aspartate 1-decarboxylase activity is as described above. For example, the unmodified microorganism used as the target strain for comparing the presence or absence of increased beta-alanine and / or beta-alanine-derived compound production capacity may be the wild-type Corynebacterium glutamicum ATCC13032 strain.

[0053] A microorganism in which the activity of aspartate 1-decarboxylase is enhanced may be a microorganism into which aspartate 1-decarboxylase derived from confused flour beetle has been introduced, and such microorganism may have 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 the other 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 mutations that increase the production of beta-alanine and / or beta-alanine-derived compounds, and the location of the mutation and / or the gene and / or protein to which it is mutated may be any type that increases the production of beta-alanine and / or beta-alanine-derived compounds. The recombinant cell may be any cell capable of transformation.

[0055] The aforementioned 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 pantothenate), malonate semialdehyde, malonate, acetylene-monocarboxylate, etc., but is not limited thereto.

[0056] As an example, microorganisms (or strains, recombinant cells) with improved (increased) beta-alanine and / or beta-alanine-derived compound production ability (or production volume) are compared to the pre-mutation parent strain, unmodified microorganisms, or microorganisms into which aspartate 1-decarboxylase from other microorganisms has been introduced, with beta-alanine and / or beta-alanine-derived compound production ability increasing by approximately 10%, 20%, 30%, 50%, 100%, 200%, 300%, 400%, and 500%. The increase may be % or more, approximately 600% or more, approximately 700% or more, approximately 800% or more, approximately 900% or more, approximately 1,000% or more, approximately 1,500% or more, approximately 2,000% or more, approximately 2,500% or more, or approximately 3,000% or more. For example, the increase may be approximately 33.3% or more, approximately 100% or more, approximately 175% or more, approximately 243.75% or more, approximately 266.6% or more, approximately 300% or more, approximately 450% or more, approximately 500% or more, approximately 587.5% or more, approximately 700% or more, or approximately 2,650% or more.

[0057] In other examples, microorganisms (or strains, recombinant cells) with increased production capacity (or yield) showed beta-alanine and / or beta-alanine-derived compound production capacity (or yield) of approximately 1.1 times, 1.3 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, and 8 times compared to the pre-mutation parent strain, unmodified microorganisms, or microorganisms into which aspartate 1-decarboxylase from other microorganisms had been introduced. The above figures may be approximately 9 times or more, approximately 10 times or more, approximately 15 times or more, approximately 20 times or more, approximately 25 times or more, or approximately 30 times or more (the upper limit is not particularly restricted and may be, for example, approximately 1,000 times or less), and may also be, but are not limited to, increases of approximately 1.3 times or more, approximately 2 times or more, approximately 2.75 times or more, approximately 3.43 times or more, approximately 3.6 times or more, approximately 4 times or more, approximately 5.5 times or more, approximately 6 times or more, approximately 6.8 times or more, approximately 8 times or more, or approximately 27.5 times or more.

[0058] In other examples, microorganisms (or strains, recombinant cells) with increased production capacity (or yield) showed beta-alanine and / or beta-alanine-derived compound production capacity (or yield) of approximately 0.5 g / L or more, approximately 1 g / L or more, approximately 1.5 g / L or more, approximately 2.0 g / L or more, approximately 2.5 g / L or more, approximately 3 g / L or more, approximately 3.5 g / L or more, approximately 4 g / L or more, approximately 4.5 g / L or more, approximately 5 g / L or more, approximately 5.5 g / L or more, and approximately 6 g / L. The above amounts may be approximately 7 g / L or more, approximately 8 g / L or more, approximately 9 g / L or more, approximately 10 g / L or more, approximately 15 g / L or more, approximately 20 g / L or more, approximately 25 g / L or more, or approximately 30 g / L or more (the upper limit is not particularly restricted and may be, for example, approximately 100 g / L or less), and may also be increased by approximately 0.6 g / L or more, approximately 1.2 g / L or more, approximately 1.8 g / L or more, approximately 2 g / L or more, approximately 2.4 g / L or more, approximately 3.5 g / L or more, approximately 3.9 g / L or more, approximately 4 g / L or more, approximately 4.5 g / L or more, approximately 4.7 g / L or more, or approximately 5.3 g / L or more, but are not limited to these.

[0059] More specifically, the microorganisms (or strains, recombinant cells) with increased production capacity (or yield) showed beta-alanine and / or beta-alanine-derived compound production capacity (or yield) of approximately 33.3%, 100%, 175%, 243.75%, 266.6%, 300%, 450%, 500%, and 587% of the pre-mutation parent strain, unmodified microorganisms, or microorganisms into which aspartate 1-decarboxylase from other microorganisms had been introduced. The increase may be 0.5%, approximately 700%, or approximately 2,650% (or approximately 1.3 times, approximately 2 times, approximately 2.75 times, approximately 3.43 times, approximately 3.6 times, approximately 4 times, approximately 5.5 times, approximately 6 times, approximately 6.8 times, approximately 8 times, or approximately 27.5 times; or approximately 0.6 g / L, approximately 1.2 g / L, approximately 1.8 g / L, approximately 2 g / L, approximately 2.4 g / L, approximately 3.5 g / L, approximately 3.9 g / L, approximately 4 g / L, approximately 4.5 g / L, approximately 4.7 g / L, approximately 5.3 g / L).

[0060] The term "about" includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value that follows the term "about," such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc.

[0061] For example, the microorganism in which the activity of aspartate 1-decarboxylase is enhanced may be a microorganism of the genus Corynebacterium sp. The aforementioned Corynebacterium species include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. It may also be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and / or Corynebacterium flavescens.

[0062] Another aspect of this application provides a method (or method of production) for beta-alanine and / or beta-alanine-derived compounds, comprising the step of culturing the microorganism of this application in a culture medium.

[0063] The method for producing beta-alanine and / or beta-alanine-derived compounds according to this application may include the step of culturing the microorganism of this application in a culture medium. The microorganism, beta-alanine-derived compounds, etc., of this application are as described above.

[0064] In this application, “cultivation” means growing a microorganism with enhanced aspartate 1-decarboxylase activity, such as the Corynebacterium glutamicum strain, under appropriately controlled environmental conditions. The cultivation process described in this application can be carried out using suitable culture media and cultivation conditions known to the art. Such a cultivation process can be readily adapted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0065] In this application, “culture medium” means a mixture mainly composed of nutrients necessary for culturing microorganisms with enhanced aspartate 1-decarboxylase activity, such as the Corynebacterium glutamicum strain, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, any culture medium and other culture conditions used for culturing the microorganisms of this application can be used without special restrictions as long as they are culture media used for culturing ordinary microorganisms, but the microorganisms of this application can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0066] Specifically, the culture medium for the microorganisms of this application, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0067] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, sugarcane meal, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0068] The nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0069] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these methods.

[0070] Furthermore, during the cultivation of the microorganisms of this application, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in an appropriate manner. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or to maintain an anaerobic and microaerobic state without injecting gas, or by injecting nitrogen, hydrogen, or carbon dioxide gas, although this is not limited to the above.

[0071] In the culture method described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0072] Beta-alanine and / or beta-alanine-derived compounds produced by the culture described in this application may be secreted into the culture medium or remain in the cells.

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

[0074] The method for producing beta-alanine and / or beta-alanine-derived compounds according to this application may additionally include a step of recovering beta-alanine and / or beta-alanine-derived compounds from the culture medium (the culture medium in which the culture was performed) or microorganisms (e.g., Corynebacterium strains). The recovery step may be additionally included after the culture step.

[0075] The aforementioned recovery may involve collecting the target 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 for culturing microorganisms as described in this application. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods can be used to recover the target beta-alanine and / or beta-alanine-derived compounds from the culture medium or microorganisms using a suitable method known in the art.

[0076] Furthermore, the method for producing beta-alanine and / or beta-alanine-derived compounds of this application may additionally include a purification step. The purification can be carried out using a suitable method known in the art. For example, if the method for producing beta-alanine and / or beta-alanine-derived compounds of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, in any order, or simultaneously or integrated into a single step, but are not limited thereto.

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

[0078] The compositions of this application may further contain any suitable excipients commonly used in compositions for the production of beta-alanine and / or beta-alanine-derived compounds, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

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

[0080] Other aspects of this application provide uses for the microorganism of this application for the production of beta-alanine and / or beta-alanine-derived compounds or for the manufacture of compositions for the production of beta-alanine and / or beta-alanine-derived compounds. The microorganism of this application, beta-alanine and / or beta-alanine-derived compounds or compositions for the production of beta-alanine and / or beta-alanine-derived compounds are as described above. [Effects of the Invention]

[0081] This application provides a microorganism with enhanced activity of aspartate 1-decarboxylase derived from confused flour beetle, the microorganism exhibiting excellent ability to produce beta-alanine and / or beta-alanine-derived compounds. [Modes for carrying out the invention]

[0082] The present invention will be described more specifically below with reference to the following embodiments. However, these are merely illustrative examples of the present invention, and the scope of the present invention is not limited by these embodiments. [Examples]

[0083] Example 1. Aspertate 1-decarboxylase gene search and selection Microbial aspartate 1-decarboxylase (or PanD) can be classified into two types: one that is active while being expressed, and another that is activated by a PanD regulatory factor (PanM). Therefore, we conducted a gene search for microorganisms that possess both aspartate 1-decarboxylase requiring a PanM regulatory factor and the active form of aspartate 1-decarboxylase.

[0084] Candidate genes and microorganisms possessing aspartate 1-decarboxylase were selected based on organisms with aspartate 1-decarboxylase in prokaryotic and eukaryotic cell systems. Aspartate 1-decarboxylase derived from microorganisms with a biosafety level of 1 was selected as shown in Table 1.

[0085] [Table 1]

[0086] Example 2. Production of Corynebacterium microorganisms into which aspartate 1-decarboxylase derived from exogenous microorganisms has been introduced. After extracting the genomes of the acquired microorganisms, PCR was performed on DNA fragments encoding aspartate 1-decarboxylase using the templates, with the primer sequences in Table 1 (SEQ ID NOs. 1 and 2, SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 11 and 12, SEQ ID NOs. 13 and 14, SEQ ID NOs. 15 and 16). PCR was performed using PfuUltra™ high-reliability DNA polymerase (Stratagene), with PCR conditions of 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 from each microorganism was obtained. In addition, PCR was performed on DNA fragments encoding PanD regulators (PanM) using the genomes of Escherichia coli and Serratia rubidaea microorganisms, with the primer sequences in Table 1 (SEQ ID NOs. 3 and 4, SEQ ID NOs. 9 and 10). PCR was performed using PfuUltra™ high-reliability DNA polymerase (Stratagene), with PCR conditions of 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, PanD regulators from Escherichia coli and Serratia rubidaea were obtained. To obtain the lysC promoter from Corynebacterium glutamicum, the genomic DNA of Corynebacterium glutamicum was used as a template, and the promoter was PCR using primers 17 and 18 in the same manner as described above to obtain the DNA fragment. Plasmids were obtained by cloning the pECCG117 vector (Registered Patent No. 10-0057684 of the Republic of Korea), which had been treated with restriction enzyme BamHI and then heat-treated at 65°C for 20 minutes, and the DNA fragment using the manual provided with the TaKaRa Infusion Cloning Kit. The vector name and information of the introduced gene are shown in Table 1.

[0087] Six types of vectors were produced and used to transform Corynebacterium glutamicum ATCC13032 into strains expressing exogenous aspartate 1-decarboxylase (PanD) via electroporation.

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

[0089] <Production culture medium> Glucose 10%, Yeast extract 0.4%, Ammonium sulfate 1.5%, Monopotassium phosphate 0.1%, Magnesium sulfate heptahydrate 0.05%, Iron sulfate heptahydrate 10mg / l, Manganese sulfate monohydrate 6.7mg / l, Biotin 50μg / l, Thiamine HCl 100μg / l, pH 7.2

[0090] [Table 2]

[0091] As a result, as shown in Table 2 above, the parent strain, Corynebacterium glutamicum ATCC13032, did not produce beta-alanine, while the strain ATCC13032 pECCG117-panD(CG), which expresses Corynebacterium glutamicum aspartate 1-decarboxylase, produced approximately 1.2 g / L of beta-alanine. Among the strains expressing aspartate 1-decarboxylase derived from exogenous microorganisms, ATCC13032 pECCG117-panD(TC), which expresses aspartate 1-decarboxylase derived from confused flour beetle (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 this invention, aspartate 1-decarboxylase derived from confused flour beetle can produce beta-alanine more efficiently.

[0093] In this example, the ATCC13032 pECCG117-panD(TC) strain (named Corynebacterium glutamicum CV03-5003), which was confirmed to have the best beta-alanine production ability, was deposited with the Korea Microbial Conservation Center located in Hongje-dong, Seodaemun-gu, Seoul, South Korea on November 23, 2021, and was assigned the deposit number KCCM13076P.

[0094] Example 4. Investigation of beta-alanine-derived compound production capacity of Corynebacterium microorganisms expressing aspartate 1-decarboxylase derived from exogenous microorganisms. To confirm the ability of the aspartate 1-decarboxylase of the confused flour beetle, as confirmed in Example 3, to produce beta-alanine-derived compounds (pantothenic acid), microorganisms with enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase (or PanB protein) were prepared.

[0095] First, a vector was prepared to delete the panB gene present in the parent strain. PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, with primers number 28 and 29 and number 30 and 31. The 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 and downstream of the panB gene were obtained, respectively. The amplification products were purified using QIAGEN's PCR Purification Kit and used as insertion DNA fragments for vector preparation.

[0096] The pDCM2_ΔpanB vector, which deletes the panB gene on a chromosome, was fabricated by cloning the pDCM2 (Registered Patent No. 2278000 in the Republic of Korea) vector, which was treated with restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, with 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) in a molar concentration (M) of 2:1:1 using the manual provided with the TaKaRa Infusion Cloning Kit.

[0097] To prepare the panB gene derived 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 the following steps 30 times: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, resulting in a 795 bp DNA fragment. To obtain the lysC promoter derived from Corynebacterium glutamicum, the promoter was obtained by PCR using primers 34 and 35 with Corynebacterium glutamicum genomic DNA as a template, in the same manner as in the previous example. The pDCM2_ΔpanB::panB(EC) vector for introducing the panB gene from E. coli onto the chromosome was prepared by cloning the pDCM2_ΔpanB vector, which had been treated with restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, with the obtained DNA fragment in a molar concentration (M) of 2:1:1, using the manual provided with the TaKaRa Infusion Cloning Kit.

[0098] The prepared vector pDCM2_ΔpanB::panB(EC) was transformed into Corynebacterium glutamicum ATCC13032 via electroporation, and strains with E. coli-derived panB introduced onto the chromosome (ΔpanB::panB(EC)) were obtained through a secondary crossover process. The presence or absence of 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. Specifically, primary determination was performed by selecting strains that were amplified by primer combinations corresponding to E. coli panB (sequences 36 and 35 and sequence numbers 37 and 32), and secondary confirmation was performed by analyzing the panB sequences of the selected strains using the primer combinations of sequence numbers 36 and 37.

[0099] After introducing the plasmid obtained in Example 1 into the ATCC13032 ΔpanB::panB(EC) strain, the parent strain and the aforementioned strain were inoculated into 250 ml corner baffle flasks containing 25 ml of production medium formed with the composition as in the example, and then cultured at 33°C for 48 hours with shaking at 200 rpm to measure pantothenic acid production capacity.

[0100] [Table 3]

[0101] As a result, as shown in Table 3 above, the parent strain, Corynebacterium glutamicum ATCC13032ΔpanB::panB(EC), produced almost no pantothenic acid, while the strain expressing Corynebacterium glutamicum aspartate 1-decarboxylase (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 exogenous microorganisms, the strain expressing aspartate 1-decarboxylase derived from Tribolium castaneum, ATCC13032 ΔpanB::panB(EC) pECCG117-panD(TC), showed the highest pantothenic acid productivity at 5.5 g / L.

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

[0103] From the above description, those skilled in the art in the field to which the present invention pertains should understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts described below rather than from the above detailed description. [Accession Number]

[0104] Depository name: Korea Microbial Conservation Center Accession number: KCCM13076P Date of acceptance: 20211123 TIFF0007836412000004.tif206168

Claims

1. A Corynebacterium microorganism that produces beta-alanine or a beta-alanine-derived compound, into which aspartate 1-decarboxylase derived from the confused flour beetle (Tribolium castaneum) or a polynucleotide encoding it has been introduced, The aspertate 1-decarboxylase derived from the confused flour beetle (Tribolium castaneum) contains an amino acid sequence that has at least 99.3% identity with the amino acid sequence represented by Sequence ID No.

27. The Corynebacterium microorganisms described above have increased production capacity for beta-alanine or beta-alanine-derived compounds compared to the parent strain or wild type in which the aspartate 1-decarboxylase or the polynucleotide encoding it has not been introduced. The aforementioned microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

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 contains the amino acid sequence represented by SEQ ID NO:

27.

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

38.

5. A method for producing beta-alanine or a beta-alanine-derived compound, comprising the step of culturing a Corynebacterium microorganism into which aspartate 1-decarboxylase derived from confused flour beetle or a polynucleotide encoding it has been introduced in a culture medium, The aspertate 1-decarboxylase derived from the confused flour beetle (Tribolium castaneum) contains an amino acid sequence that has at least 99.3% identity with the amino acid sequence represented by Sequence ID No.

27. The Corynebacterium microorganisms described above have increased production capacity for beta-alanine or beta-alanine-derived compounds compared to the parent strain or wild type in which the aspartate 1-decarboxylase or the polynucleotide encoding it has not been introduced. The aforementioned Corynebacterium microorganism is Corynebacterium glutamicum, and the method for producing it.

6. The production method according to claim 5, further comprising the step of recovering beta-alanine or a beta-alanine-derived compound from the culture medium or microorganism obtained by the culture.

7. A composition for the production of beta-alanine or beta-alanine-derived compounds, comprising a Corynebacterium microorganism into which aspartate 1-decarboxylase derived from confused flour beetle or a polynucleotide encoding it has been introduced, The aspertate 1-decarboxylase derived from the confused flour beetle (Tribolium castaneum) contains an amino acid sequence that has at least 99.3% identity with the amino acid sequence represented by Sequence ID No.

27. The Corynebacterium microorganisms described above have increased production capacity for beta-alanine or beta-alanine-derived compounds compared to the parent strain or wild type in which the aspartate 1-decarboxylase or the polynucleotide encoding it has not been introduced. The aforementioned Corynebacterium microorganism is Corynebacterium glutamicum, and this is a composition for production.

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