Microorganisms with enhanced aspartate 1-decarboxylase activity derived from tribolium cavaleriei and uses thereof

Abstract

The present application provides: a microorganism which is derived from tribolium castaneum and has enhanced aspartate 1-decarboxylase activity; a composition for producing beta-alanine and / or a beta-alanine derivative compound, said composition comprising said microorganism; and a method for producing beta-alanine and / or a beta-alanine derivative compound, the method comprising a step of culturing the microorganism. According to the present invention, the production capacity of beta-alanine and / or a beta-alanine-derived compound is excellent.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0034234 filed on March 18, 2022, and all contents disclosed in the corresponding Korean patent application document are incorporated as a part of this specification.

[0003] The present invention provides a microorganism having enhanced aspartate 1-decarboxylase activity derived from Tribolium castaneum, a composition for producing β-alanine and / or β-alanine-derived compounds comprising the microorganism, and a method for producing β-alanine and / or β-alanine-derived compounds, comprising culturing the microorganism. Background Art

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

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

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

[0007] Prior art

[0008] Patent Literature

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

[0010] Technical issues

[0011] The present application provides a microorganism having enhanced aspartate 1-decarboxylase activity and enhanced production capacity of β-alanine and / or β-alanine-derived compounds.

[0012] The present application provides a composition for producing β-alanine and / or β-alanine-derived compounds, comprising the microorganism.

[0013] The present application provides a method for producing β-alanine and / or β-alanine derivative compounds, comprising culturing the microorganism in a culture medium.

[0014] The present application provides a use of a microorganism for producing β-alanine and / or β-alanine derivative compounds.

[0015] The present application provides a use of a microorganism for preparing a composition for producing β-alanine and / or β-alanine derivative compounds.

[0016] Technical Solution

[0017] The present application relates to a method for producing and / or enhancing the production capacity of aspartate and / or aspartate-1-decarboxylase for β-alanine-derived compounds, wherein the aspartate-1-decarboxylase, for example, is derived from Tribolium castaneum.

[0018] One aspect provides a microorganism (or strain, recombinant cell) having enhanced aspartate 1-decarboxylase activity.

[0019] In the present application, aspartate 1-decarboxylase (or PanD protein) may refer to a protein having aspartate 1-decarboxylase activity. Aspartate 1-decarboxylase may be derived from Tribolium castaneum, Red flourbeetle, Escherichia coli, Bacillus subtilis, Serratia rubidaea, Corynebacterium glutamicum or Pseudomonas sp. bacteria, but is not limited thereto. In one embodiment, aspartate 1-decarboxylase may be derived from Tribolium castaneum, and the sequence of the aspartate 1-decarboxylase derived from Tribolium castaneum may be obtained from the known database NCBI, and for example, it may be represented by NCBI reference sequence NP_001096055.1. In one embodiment, the aspartate 1-decarboxylase derived from Tribolium castaneum may have or include, or consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 27. Specifically, the aspartate 1-decarboxylase may consist of a polypeptide described as the amino acid sequence of SEQ ID NO: 27.

[0020] In one embodiment, the aspartate 1-decarboxylase protein can 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 to the amino acid sequence of SEQ ID NO: 27, or comprise or consist of the sequence, but is not limited thereto. In addition, as long as it is an amino acid sequence having such homology or identity and showing an effect corresponding to aspartate 1-decarboxylase, aspartate 1-decarboxylase may also include a variant having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added. For example, the addition or deletion of a sequence that does not change the activity of aspartate 1-decarboxylase at the N-terminus, C-terminus and / or inside the amino acid sequence may be a naturally occurring mutation, silent mutation or conservative substitution.

[0021] A "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0022] In the present application, aspartate 1-decarboxylase can be a polypeptide having aspartate 1-decarboxylase activity encoded by an aspartate 1-decarboxylase gene. The aspartate 1-decarboxylase gene can be derived from Tribolium castaneum, Escherichia coli, Bacillus subtilis, Serratia rubrum, Corynebacterium glutamicum or Pseudomonas bacteria, but is not limited thereto. In one embodiment, the aspartate 1-decarboxylase gene can be derived from Tribolium castaneum. The sequence of the aspartate 1-decarboxylase gene derived from Tribolium castaneum can be obtained from the known database NCBI, and for example, it can be represented by NCBI reference sequence: NM_001102585.1. In one embodiment, the aspartate 1-decarboxylase gene derived from Tribolium castaneum can have or include the nucleic acid sequence of SEQ ID NO:38, or be composed of the nucleic acid sequence, or be substantially composed of the nucleic acid sequence. Specifically, the aspartate 1-decarboxylase gene can be composed of the nucleic acid sequence of SEQ ID NO:38. In one embodiment, the aspartate 1-decarboxylase gene may comprise 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 SEQ ID NO:38, or consist of said sequence.

[0023] In the present application, a polynucleotide or polypeptide "having or containing a specific nucleic acid sequence (base sequence) or amino acid sequence, or consisting of the sequence, or substantially consisting of the sequence" may refer to a polynucleotide or polypeptide that substantially contains a specific nucleic acid sequence (base sequence) or amino acid sequence, and it may be interpreted as a "substantially equivalent sequence" (or not excluding the mutation) applied to the specific nucleic acid sequence (base sequence) or amino acid sequence within the scope of maintaining the original function and / or required function of the polynucleotide or polypeptide. In one embodiment, a polynucleotide or polypeptide "having or comprising a specific nucleic acid (base sequence) or amino acid sequence, consisting of the sequence, or substantially consisting of the sequence" may mean that the polynucleotide or polypeptide (i) substantially comprises the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of 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, or substantially comprises the specific nucleic acid sequence (base sequence) or amino acid sequence, and maintains the original function and / or the desired function. In one embodiment, the desired function may refer to a function of increasing (enhancing) or imparting the production capacity of β-alanine and / or β-alanine-derived compounds of the microorganism.

[0024] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or base sequences, and can be expressed as a percentage. The terms, homology and identity are often used interchangeably.

[0025] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard array algorithms, and can be used together with the default gap penalty set up by the program used. Basically, sequences of homology or identity can hybridize with the whole or part of the sequence under medium or high stringency conditions usually. Obviously, hybridization also includes hybridization of polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotides.

[0026] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program, such as using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. In addition, the Needlean-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] et al., J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ed.] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information Database can be used to determine homology, similarity or identity.

[0027] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48: 443, as known in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program can be defined as the value obtained by dividing the total number of symbols in the shorter of the two sequences by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a binary comparison matrix (containing identical values ​​of 1 and different values ​​of 0) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol of each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0028] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and may be a microorganism comprising a genetic modification for producing a targeted polypeptide, protein or product (e.g., aspartate 1-decarboxylase), wherein the microorganism is a microorganism in which the specific mechanism is enhanced or weakened due to, for example, the insertion of a foreign gene or the enhancement or weakening of the activity of an endogenous gene.

[0029] In the present application, the term "enhancing" of polypeptide activity refers to that the activity of polypeptide increases compared with endogenous activity.Enhancing can be used interchangeably with terms such as activation, up-regulation, overexpression, increase.Herein, activation, up-regulation, overexpression and increase can include showing an activity that does not exist originally, or show an activity that is improved compared with the activity before endogenous activity or modification." Endogenous activity" refers to the activity of a specific polypeptide initially possessed by a parent strain or unmodified microorganism before the proterties change due to a genetic mutation caused by natural or artificial factors.This can be used interchangeably with "activity before modification".Compared with endogenous activity, polypeptide activity "enhancing", "up-regulation", "overexpression" or "increase" refers to the activity and / or concentration (expression level) of a specific polypeptide initially possessed by a parent strain or unmodified microorganism before the proterties change, and polypeptide activity improves.

[0030] Enhancement can be achieved by introducing exogenous polypeptides or can be to enhance the activity and / or concentration (expression level) of endogenous polypeptides. Whether the polypeptide activity is enhanced can be confirmed by the increase in the degree of activity, the expression level of the corresponding polypeptide or the amount of product released from the corresponding polypeptide.

[0031] For the enhancement of polypeptide activity, various methods known in the art can be applied, and there is no restriction, as long as the activity of the target polypeptide can be enhanced compared with the activity of the microorganism before modification. Specifically, genetic engineering and / or protein engineering known to those skilled in the art can be used, which are conventional methods of molecular biology, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0032] Specifically, the enhancement of the polypeptide of the present application can be:

[0033] 1) The cell copy number of the polynucleotide encoding the polypeptide increases;

[0034] 2) Replace the gene expression regulatory region encoding the polypeptide on the chromosome with a sequence with strong activity;

[0035] 3) modifying the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide;

[0036] 4) Modify the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

[0037] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide of a polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

[0038] 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding a polypeptide;

[0039] 7) codon-optimized polynucleotides encoding polypeptides;

[0040] 8) by analyzing the tertiary structure of the polypeptide and selecting modification or chemical modification of the exposed sites; or

[0041] 9) A combination of at least two selected from the above 1) to 8), but not limited thereto.

[0042] More specifically, the increase in the number of copies of the polynucleotide encoding the polypeptide in the cell of 1) above can be achieved by introducing a vector that can replicate and function independently of the host, wherein the polynucleotide encoding the corresponding polypeptide is operably linked. In addition, it can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited thereto. The vector is as described above.

[0043] In the above 2), replacing the gene expression control region (or expression control sequence) encoding the polypeptide on the chromosome with a sequence having strong activity can be, for example, a mutation of the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, to further enhance the activity of the expression control region, or replace it with a sequence having stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, etc. As an example, the original promoter can be replaced with a strong promoter, but is not limited thereto.

[0044] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 1,058,4338 B2), O2 promoter (U.S. Patent No. 1,027,3491 B2), tkt promoter, yccA promoter, etc., but are not limited to these.

[0045] In 3) above, the modification of the start codon or 5'-UTR base sequence of the gene transcriptome encoding the polypeptide may be, for example, replacement with a base sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0046] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide with an improved amino acid sequence or polynucleotide sequence with stronger activity, thereby enhancing the activity of the polypeptide, but not limited to this. The polynucleotide can be specifically inserted into the chromosome for replacement by homologous recombination, but not limited to this. The vector used then can also include a selection marker for confirming chromosome insertion. The selection marker is as described above.

[0047] In the above 6), the introduction of an exogenous polypeptide exhibiting polypeptide activity can be the introduction of an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The source or sequence of the exogenous polynucleotide is not limited, as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be appropriately selected by a person skilled in the art by a known transformation method, and by expressing the introduced polynucleotide in a host cell, a polypeptide can be produced and its activity can be increased.

[0048] The codon-optimized polypeptide encoding polynucleotide in the above 7) may be codon-optimized so as to increase the transcription or translation of an endogenous polynucleotide in a host cell, or codon-optimized so as to optimize the transcription and translation of an exogenous polynucleotide in a host cell.

[0049] In the above 8), by analyzing the tertiary structure of the polypeptide and selecting the modification or chemical modification of the exposed site, the template protein candidate can be determined according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed, based on which the structure is determined and the exposed site to be modified or chemically modified is selected, and it is transformed or modified.

[0050] Such enhancement of polypeptide activity can be based on the activity or concentration of the polypeptide expressed in the wild-type or modified microbial strain, an increase in the activity or concentration expression level of the corresponding polypeptide, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0051] In the present application, the term "weakening" of a polypeptide is a concept that includes reduced activity or no activity compared to endogenous activity. Weakening can be used interchangeably with terms such as inactivation, deletion, downregulation, reduction, decrease, attenuation, etc. Weakening can also include the situation where the activity of the polypeptide itself is reduced or eliminated due to mutations of polynucleotides encoding polypeptides (or proteins, such as aspartate 1-decarboxylase) compared to the activity of the polypeptide possessed by the original microorganism; the situation 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 expression of the gene encoding the polynucleotide or inhibition of translation into polypeptides; the situation where the polynucleotide is not expressed at all; and / or the situation where the polypeptide is not active even if the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait is changed when the trait is changed due to genetic mutations caused by natural or artificial factors. Endogenous activity can be used interchangeably with "activity before modification". The activity of the polypeptide is "inactivated, deleted, reduced, downregulated, decreased or attenuated" compared to the endogenous activity, meaning that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait is changed.

[0052] The weakening of the activity of this polypeptide (or protein, such as aspartate 1-decarboxylase) can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods 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.).

[0053] Specifically, attenuation of a polypeptide (or protein, such as aspartate 1-decarboxylase; hereinafter referred to as a polypeptide) can be

[0054] 1) Deletion of all or part of the gene encoding the polypeptide;

[0055] 2) modifying the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0056] 3) modifying (e.g., deletion / substitution / addition of at least one amino acid in the amino acid sequence) the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide;

[0057] 4) modifying (e.g., adding / replacing / adding at least one nucleic acid base in the nucleic acid base sequence of a polypeptide gene encoding a polypeptide modified to eliminate or weaken the activity of the polypeptide) a gene sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide;

[0058] 5) modifying the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide;

[0059] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcriptome of the gene encoding the polypeptide;

[0060] 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which the ribosome cannot attach;

[0061] 8) adding a promoter that transcribes 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); or

[0062] 9) A combination of at least two selected from 1) to 8), but not limited thereto.

[0063] For example,

[0064] Deleting all or part of the gene encoding the polypeptide in 1) above may be accomplished by removing the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polypeptide lacking some nucleotides, or replacing it with a marker gene.

[0065] In addition, the modification of the expression control region (or expression control sequence) in the above 2) may be mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having weaker activity. The expression control region includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, but is not limited thereto.

[0066] In addition, the modification of the start codon or 5'-UTR base sequence of the gene transcriptome encoding the polypeptide in 3) above may be, for example, 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.

[0067] In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of the gene can be suppressed or weakened, but is not limited thereto. A "stop codon" is a codon that does not specify an amino acid in the codon on the mRNA, notifies the end of the protein synthesis process, and acts as a signal, and generally uses UAA, UAG, and UGA as stop codons.

[0068] Regarding the introduction of antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to the transcriptome of the gene encoding the polypeptide in 6) above, reference can be made to, for example, the literature [Weintraub, H. et al., Antisense-RNA as amolecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0069] In the above 7), a sequence complementary to the Shine-Dalgarno sequence is added before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which the ribosome cannot attach, thereby making the mRNA unable to be translated or inhibiting the mRNA translation rate.

[0070] In the above 8), adding 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 produce antisense nucleotides complementary to the transcriptome of the gene encoding the polypeptide to weaken the activity.

[0071] The microorganism of the present application may be a microorganism (eg, a recombinant microorganism) modified by a vector gene, thereby enhancing the activity of aspartate 1-decarboxylase or a polynucleotide encoding the same, but is not limited thereto. The vector is as described above.

[0072] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having the ability to produce β-alanine and / or β-alanine derivative compounds or having enhanced production ability (or yield) of β-alanine and / or β-alanine derivative compounds.

[0073] The microorganism of the present application may be a microorganism that naturally does not have the ability to produce β-alanine and / or β-alanine derivative compounds by introducing or enhancing the activity of aspartate 1-decarboxylase, thereby conferring or enhancing the ability to produce β-alanine and / or β-alanine derivative compounds, or a microorganism that has the ability to produce β-alanine and / or β-alanine derivative compounds, but is not limited thereto. The microorganism that confers or enhances the ability to produce β-alanine and / or β-alanine derivative compounds may be a microorganism into which an aspartate 1-decarboxylase derived from Tribolium castaneum is introduced.

[0074] The microorganism (or strain, recombinant cell) having enhanced production capacity (or yield) of β-alanine or β-alanine-derived compounds, or having the production capacity of β-alanine or β-alanine-derived compounds can mean that, compared with an unmodified microorganism, a cell before recombination, a parent strain, a wild-type strain and / or a microorganism into which an aspartate 1-decarboxylase derived from other microorganisms is introduced, the microorganism (or strain, recombinant cell) has enhanced production capacity of β-alanine or β-alanine-derived compounds, or the microorganism (or strain, recombinant cell) is endowed with β-alanine or β-alanine-derived compound production capacity different from that of an unmodified microorganism, a cell before recombination, a parent strain and / or a wild-type strain that does not have the production capacity of β-alanine or β-alanine-derived compounds.

[0075] Compared with the microorganism before enhancing (that is, the unmodified microorganism of the same kind), the microorganism with enhanced aspartate 1-decarboxylase activity according to an embodiment can have improved (increased) beta-alanine and / or beta-alanine derivative compound production capacity.In the application, "unmodified microorganism" does not exclude the bacterial strain comprising the mutation that can be naturally occurring in the microorganism, can refer to wild-type strain or natural strain itself, or the bacterial strain before changing the character caused by the gene mutation caused by natural or artificial factors.For example, unmodified microorganism can refer to the bacterial strain before the activity of aspartate 1-decarboxylase is not enhanced or enhanced according to an embodiment (the mutation that induces the activity enhancement of aspartate 1-decarboxylase is not introduced or the bacterial strain before it is introduced). "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "non-mutant strain", "unmodified strain", "microorganism that does not mutate" or "reference microorganism".Enhancing the activity of aspartate 1-decarboxylase is as described above. In one embodiment, the unmodified microorganism (which is a test strain for comparing whether the production capacity of β-alanine and / or β-alanine-derived compound is increased) may be a wild-type Corynebacterium glutamicum ATCC13032 strain.

[0076] According to one embodiment, the microorganism with enhanced aspartate 1-decarboxylase activity can be a microorganism into which an aspartate 1-decarboxylase derived from Tribolium castaneum has been introduced, and compared with a microorganism into which an aspartate 1-decarboxylase derived from another microorganism has been introduced, the microorganism can have enhanced (increased) β-alanine and / or β-alanine derivative compound production capacity. The aspartate 1-decarboxylase derived from another microorganism can be derived from Escherichia coli, Bacillus subtilis, Serratia rubrum, Corynebacterium glutamicum or Pseudomonas bacteria.

[0077] The microorganism (or strain, recombinant cell) may include mutations to further increase the production of β-alanine and / or β-alanine derivative compounds, and may include any position of the mutation and any kind of gene and / or protein subjected to the mutation without limitation, as long as it increases the production of β-alanine and / or β-alanine derivative compounds. Recombinant cells may be used without limitation, as long as they are cells capable of transformation.

[0078] The β-alanine derivative compound can be at least one selected from the group consisting of β-nitropropionic acid, β-amino-propionitrile, N-acetyl-β-alanine, L-aspartic acid, anserine, spermine, carnosine, β-alanylarginine, quinolinate, pantothenate (or pantothenic acid), malonic semialdehyde, malonate, acetylene-monocarboxylate, etc., but is not limited thereto.

[0079] As an example, a microorganism (or strain, recombinant cell) having an improved (increased) production capacity (or yield) of β-alanine and / or β-alanine-derived compounds may have an increase of about 10% 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 80% or more, about 900% or more, compared to a parent strain before mutation, an unmodified microorganism, or a microorganism into which an aspartate 1-decarboxylase derived from another microorganism is introduced. , about 1000% or more, about 1500% or more, about 2,000% or more, about 2,500% or more, or about 3,000% or more of the production capacity of β-alanine and / or β-alanine-derived compounds, and in one embodiment, the microorganism (or strain, recombinant cell) can increase by 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.

[0080] In another embodiment, the microorganism (or strain, recombinant cell) with enhanced production capacity (or yield) can have 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, about 9 times or more, about 10 times or more, about 15 times or more, about 20 times or more, about 25 times or more, compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which an aspartate 1-decarboxylase derived from another microorganism is introduced. The production capacity (or yield) of β-alanine and / or β-alanine derivative compounds can be increased by about 1.3 times or more, about 2 times or more, about 2.75 times or more, about 3.43 times or more, about 3.6 times or more, about 4 times or more, about 5.5 times or more, about 6 times or more, about 6.8 times or more, about 8 times or more, or about 27.5 times or more, but are not limited thereto.

[0081] In another embodiment, the microorganism (or strain, recombinant cell) with enhanced productivity (or yield) can have 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, 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 11 g / L or more, about 12 g / L or more, about 13 g / L or more, about 14 g / L or more, about 15 g / L or more, about 16 g / L or more, about 17 g / L or more, about 18 g / L or more, about 19 g / L or more, about 20 g / L or more, about 21 g / L or more, about 22 g / L or more, about 23 g / L or more, about 24 g / L or more, about 25 g / L or more, about 26 g / L or more, about 27 g / L or more, about 28 g / L or more, about 29 g / L or more, about 30 g / L or more, about 31 g / L or more, about 32 g / L or more, about 33 g / L or more, about 34 g / L or more, about 35 g / L or more, about 36 g / L or more, about 37 g / L or more, about 38 g / L or more, about 39 g / L or more, about 40 g / L or more, about 41 g / L or more In one embodiment, the microorganism (or strain, recombinant cell) can increase the production capacity (or yield) of β-alanine and / or β-alanine derivative compounds by 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, about 5.3 g / L or more, but is not limited thereto.

[0082] More specifically, the microorganism (or strain, recombinant cell) with enhanced production capacity (or yield) can be increased by about 33.3%, about 100%, about 175%, about 243.75%, about 266.6%, about 300%, about 450%, about 500%, about 587.5%, about 700% or about 2,650% (or about 1.3 times, about 2 times, about 3 times, about 4 times, about 6 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 21 times, about 26 times, about 28 times, about 29 times, about 30 times, about 30 times, about 45 times, about 500%, about 587.5%, about 700% or about 2,650% (or about 1.3 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 15 times, about 2 The production capacity (or output) of β-alanine and / or β-alanine derivative compounds may be increased by about 2.75 times, about 3.43 times, about 3.6 times, about 4 times, about 5.5 times, about 6 times, about 6.8 times, about 8 times or about 27.5 times; 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), but is not limited to this.

[0083] The term "about" includes all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within the range equivalent to or similar to the value after the term about, but is not limited thereto.

[0084] In one embodiment, the microorganism having enhanced aspartate 1-decarboxylase activity may be a microorganism of the genus Corynebacterium. The Corynebacterium microorganism can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris and / or Corynebacterium flavescens.

[0085] Another aspect of the present application provides a method for producing (or preparing) β-alanine and / or β-alanine derivative compounds, comprising culturing the microorganism of the present application in a culture medium.

[0086] The method for producing β-alanine and / or β-alanine derivative compounds of the present application may include culturing the microorganism of the present application in a culture medium. The microorganism, β-alanine derivative compounds, etc. of the present application are as described above.

[0087] In the present application, "cultivation" refers to growing a microorganism containing the aspartate 1-decarboxylase activity of the present application under appropriately adjusted environmental conditions. The cultivation process of the present application can be carried out according to appropriate culture media and culture conditions known in the art. According to the selected strain, those skilled in the art can easily adjust and use the cultivation process. Specifically, the cultivation can be batch, continuous and / or batch feeding, but is not limited to this.

[0088] In the present application, "culture medium" refers to a material that mixes the nutrients required for culturing the microorganism with enhanced aspartate 1-decarboxylase activity of the present application as the main component, and provides nutrients and growth factors, etc., including water that is essential for survival and growth. Specifically, a culture medium and other culture conditions for culturing the microorganism of the present application can be used, as long as it is a culture medium for culturing common microorganisms without particular restrictions, but the microorganism of the present application can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions by adjusting temperature, pH, etc.

[0089] Specifically, the culture medium of the microorganisms used in the present application, for example, the culture medium of Corynebacterium strains can be found in the document ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0090] In the present application, as a carbon source, it can include: carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residues and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted with reducing sugars) can be used, and other carbon sources of appropriate amounts can be used differently without limitation. These carbon sources can be used alone or in combination of at least two, but are not limited thereto.

[0091] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium citrate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., organic nitrogen sources such as amino acids including glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrate, fish or its degradation product, defatted soybean cake or its degradation product, etc. can be used alone or in combination of at least two, but are not limited thereto.

[0092] As a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or a sodium salt corresponding thereto may be included. As an inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition to these, amino acids, vitamins and / or appropriate precursors may be included. These components or precursors may be added in batches or in a continuous manner. However, this is not limited thereto.

[0093] In addition, by appropriate method during the cultivation of the microorganism of the present application, in substratum, add compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid etc., regulate the pH of substratum.In addition, during the cultivation, defoamer such as fatty acid polyglycol ester can be used to suppress foaming.In addition, in order to maintain the aerobic state of substratum, oxygen or oxygen-containing gas can be injected into substratum, or in order to keep anaerobic and micro-aerobic state, gas can not be injected or nitrogen, hydrogen or carbon dioxide can be injected, but is not limited to this.

[0094] The culture temperature in the culture of the present application may be maintained at 20° C.-45° C., specifically 25° C.-40° C., and the culture may be performed for about 10-160 hours, but is not limited thereto.

[0095] The β-alanine and / or β-alanine derivative compound produced by the culture of the present application may be released into the medium or retained in the cells.

[0096] The method for producing β-alanine and / or β-alanine derivative compounds of the present application may further include, for example, preparing the microorganism (strain) of the present application, preparing a culture medium for culturing the microorganism, or a combination thereof (in any order) before culturing.

[0097] The production method of β-alanine and / or β-alanine derivative compounds of the present application may further include recovering β-alanine and / or β-alanine derivative compounds from a culture medium (a culture medium in which culture is performed) or a microorganism (e.g., a Corynebacterium strain) according to the culture. The recovery may further include after the culture.

[0098] Recovery can be based on the culture method of the microorganism of the present application, such as batch, continuous or batch feeding culture method, etc., by using appropriate methods known in the art to collect the target β-alanine and / or β-alanine derivative compounds. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out method), extraction, ultrasonic crushing, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or a combination of these methods, and using appropriate methods known in the art, the target β-alanine and / or β-alanine derivative compounds can be recovered from the culture medium or microorganism.

[0099] In addition, the production method of β-alanine and / or β-alanine derivative compounds of the present application may also include a purification step. Suitable methods known in the art may be used for purification. In one embodiment, when the method for producing β-alanine and / or β-alanine derivative compounds of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed by integrating into one step, without limitation thereto.

[0100] Another aspect of the present application provides a composition for producing β-alanine and / or β-alanine derivative compounds, which comprises the microorganism of the present application, a culture medium in which the microorganism is cultured, or a combination thereof.

[0101] The composition of the present application may also include any suitable excipients commonly used in the composition for producing β-alanine and / or β-alanine derivative compounds, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or tonicity agents, etc., but are not limited thereto.

[0102] In the composition of the present application, the microorganism (strain), culture medium, β-alanine derivative compound, etc. are as described in other aspects above.

[0103] Another aspect of the present application provides a use of the microorganism of the present application for producing β-alanine and / or β-alanine derivative compounds or preparing a composition for producing β-alanine and / or β-alanine derivative compounds. The microorganism, β-alanine and / or β-alanine derivative compounds or the composition for producing β-alanine and / or β-alanine derivative compounds of the present application are as described above.

[0104] Beneficial Effects

[0105] The present application provides a microorganism derived from Tribolium castaneum with enhanced aspartate 1-decarboxylase activity, wherein the microorganism has excellent production capacity of β-alanine and / or β-alanine-derived compounds. DETAILED DESCRIPTION

[0106] Hereinafter, the present invention will be described in more detail by the following examples. However, they are only intended to illustrate the present invention, but the scope of the present invention is not limited by these examples.

[0107] Example 1. Research and selection of aspartate 1-decarboxylase genes

[0108] Aspartate 1-decarboxylase (or PanD) derived from microorganisms is divided into two categories, that is, a form with activity when expressed and a form activated by PanD regulatory factors (PanM, PanD regulatory factors). Therefore, genetic research is carried out on microorganisms with aspartate 1-decarboxylase derived from microorganisms that require PanM regulatory factors and active aspartate 1-decarboxylase. Based on prokaryotic cells and eukaryotic cells, on the basis of organisms with aspartate 1-decarboxylase, candidate genes and microorganisms with the candidate genes are selected. Among them, aspartate 1-decarboxylase derived from microorganisms with a biosafety factor of 1 is selected, as shown in Table 1.

[0109] [Table 1]

[0110] Evaluation of microorganisms with aspartate 1-decarboxylase activity

[0111]

[0112]

[0113] Example 2. Construction of a Corynebacterium microorganism into which aspartate 1-decarboxylase derived from an exogenous microorganism is introduced

[0114] Using 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), after extracting the genome of the microorganism, it was used as a template to perform PCR on the DNA fragment encoding aspartate 1-decarboxylase. PCR was performed using PfuUltraTM High Reliability DNA Polymerase (Stratagene), and the conditions for PCR were 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 3 minutes, repeated 30 times. As a result, aspartate 1-decarboxylase derived from each microorganism was obtained. In addition, for the DNA fragment encoding the PanD regulatory factor (PanM), the genomes of the microorganisms of Escherichia coli and Serratia rubrum were used as templates, and PCR was performed using the primer sequences in Table 1 (SEQ ID NOs: 3 and 4, SEQ ID NOs: 9 and 10). PCR was performed using PfuUltraTM high reliability DNA polymerase (Stratagene), and the conditions for PCR were 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 3 minutes, repeated 30 times. As a result, PanD regulatory factors derived from Escherichia coli and Serratia rubrum were obtained. In order to obtain the lysC promoter derived from Corynebacterium glutamicum, PCR was performed using primers 17 and 18 as promoters and genomic DNA of Corynebacterium glutamicum as templates, and the method was the same as above to obtain DNA fragments. The pECCG117 (Korean Patent No. 10-0057684) vector was treated with restriction enzyme BamHI, then heat treated at 65°C for 20 minutes, and the DNA fragments were cloned using TaKaRa's Infusion cloning kit according to the manual provided to obtain plasmids, for which the vector name and the information of the introduced gene are shown in Table 1.

[0115] The six constructed vectors were transformed into Corynebacterium glutamicum ATCC 13032 by electroporation to construct strains expressing exogenous aspartate 1-decarboxylase (PanD).

[0116] Example 3. Investigation of β-alanine-producing ability of microorganisms of the genus Corynebacterium in which aspartate 1-decarboxylase derived from an exogenous microorganism is expressed

[0117] The strain obtained in Example 2 was inoculated into a 250 ml baffled flask containing 25 ml of a production medium consisting of the following components with the parent strain, and cultured at 33° C. with shaking at 200 rpm for 48 hours. The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then the supernatant was diluted to 1 / 10 with TDW (triplet distilled water), and then subjected to HPLC analysis to measure the concentration of β-alanine, and the results are shown in Table 2 below.

[0118] <Production medium>

[0119] Glucose 10%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium dihydrogen 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

[0120] [Table 2]

[0121] β-Alanine-producing ability of microorganisms expressing aspartate 1-decarboxylase derived from various microorganisms

[0122] β-Alanine concentration (g / L) ATCC13032 (wild type) 0.0 ATCC13032pECCG117-panD_panM(EC) 0.6 ATCC13032pECCG117-panD(BS) 1.8 ATCC13032pECCG117-panD_panM(SR) 0.4 ATCC13032pECCG117-panD(CG) 1.2 ATCC13032pECCG117-panD(PS) 0.3 ATCC13032pECCG117-panD(TC) 2.4

[0123] The results are shown in Table 2, which confirmed that the parent strain, Corynebacterium glutamicum ATCC 13032, did not produce β-alanine, and in the strain ATCC13032 pECCG117-panD (CG) expressing aspartate 1-decarboxylase of Corynebacterium glutamicum, about 1.2 g / L of β-alanine was produced. Among the strains expressing aspartate 1-decarboxylase derived from exogenous microorganisms, the strain ATCC13032 pECCG117-panD (TC) expressing aspartate 1-decarboxylase derived from Tribolium castaneum showed the highest β-alanine production capacity of 2.4 g / L.

[0124] The above results indicate that, among the six enzymes selected in the present invention, aspartate 1-decarboxylase derived from Tribolium castaneum can more efficiently produce β-alanine.

[0125] In this example, it was confirmed that the ATCC13032 pECCG117-panD (TC) strain (named Corynebacterium glutamicum CV03-5003) had the best β-alanine production capacity. The strain was deposited at the Korea Collection of Microorganisms in Hongje-dong, Seodaemon-gu, Seoul, South Korea on November 23, 2021, and was given the deposit number KCCM13076P.

[0126] Example 4. Investigation of the ability of a microorganism of the genus Corynebacterium to produce a β-alanine-derived compound in which aspartate 1-decarboxylase derived from an exogenous microorganism is expressed

[0127] In order to confirm the β-alanine-derived compound (pantothenic acid) production ability of the aspartate 1-decarboxylase of Tribolium castaneum confirmed in Example 3, a microorganism having enhanced 3-methyl-2-oxobutyrate methyl hydroxymethyltransferase (or PanB protein) activity was constructed.

[0128] First, a vector for deleting the panB gene present in the parent strain was constructed. Using the genomic DNA of Corynebacterium glutamicum ATCC1303 as a template, PCR was performed using primers SEQ ID NOs: 28 and 29 and SEQ ID NOs: 30 and 31. PCR was performed at 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute, repeated 30 times. As a result, a gene fragment of 1000 bp from the upper part of the panB gene and a gene fragment of 1000 bp from the lower part of the panB gene were obtained, respectively, and each amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction.

[0129] The pDCM2 (Korean Patent No. 2278000) vector was treated with the restriction endonuclease smaI, then heat-treated at 65°C for 20 minutes, and the DNA fragments (a 1000 bp gene fragment from the upper part of the panB gene and a 1000 bp gene fragment from the lower part of the panB gene) were cloned using TaKaRa's Infusion cloning kit according to the manual provided so that the molar concentration (M) was 2:1:1, thereby constructing the vector pDCM2_ΔpanB for deleting the panB gene on the chromosome.

[0130] In order to prepare the panB gene derived from Escherichia coli, the genomic DNA of Escherichia coli K12 wild-type strain (KCTC1116) was used as a template and PCR was performed using primers 32 and 33. PCR was performed by 95°C, denaturation for 30 seconds; 55°C, annealing for 30 seconds; and 72°C, polymerization for 1 minute and 30 seconds, repeated 30 times, and a DNA fragment of 795bp was obtained as a result. In order to obtain the lysC promoter derived from Corynebacterium glutamicum, the genomic DNA of Corynebacterium glutamicum was used as a template and primers 34 and 35, and PCR was performed in the same manner as in the above embodiment to obtain a DNA fragment. The pDCM2_ΔpanB vector and the obtained DNA fragment, which were treated with restriction endonuclease smaI and then heat-treated at 65°C for 20 minutes, were cloned using TaKaRa's Infusion cloning kit according to the manual provided, so that the molar concentration (M) was 2:1:1, to construct a vector pDCM2_ΔpanB::panB (EC), which was used to introduce the panB gene derived from Escherichia coli into the chromosome.

[0131] By transforming the constructed vector pDMC2_ΔpanB::panB (EC) into Corynebacterium glutamicum ATCC13032 by electroporation, and by a secondary hybridization process, strains (ΔpanB::panB (EC)) into which the panB gene derived from Escherichia coli was introduced on the chromosome were obtained respectively. The appropriate replacement of panB derived from Escherichia coli was confirmed using the following primer combination, using the MASA (mutant allele specific amplification) PCR method (Takeda et al., Hum. Mutation, 2, 112-117 (1993)). In other words, the primer combination (SEQ ID No: 36 and 35 and SEQ ID No: 37 and 32) matching the panB gene of Escherichia coli was first determined by selecting the amplified strain, and the panB gene sequence of the selected strain was secondarily confirmed by analyzing using the primer combination of SEQ ID NO: 36 and SEQ ID NO: 37.

[0132] The plasmid obtained in Example 1 was introduced into the ATCC13032ΔpanB::panB(EC) strain, and then the parent strain and the strain were respectively inoculated into a 250 ml baffled flask containing 25 ml of a production medium composed of the components of the above example, and then cultured at 33°C with shaking at 200 rpm for 48 hours to measure the pantothenic acid production capacity.

[0133] [Table 3]

[0134] Pantothenate-producing ability of microorganisms expressing aspartate 1-decarboxylase derived from various microorganisms

[0135] Pantothenic acid concentration (g / L) ATCC13032ΔpanB::panB(EC) 0.2 ATCC13032ΔpanB::panB(EC)pECCG117-panD_panM(EC) 1.0 ATCC13032ΔpanB::panB(EC)pECCG117-panD(BS) 1.6 ATCC13032ΔpanB::panB(EC)pECCG117-panD_panM(SR) 0.8 ATCC13032ΔpanB::panB(EC)pECCG117-panD(CG) 2.0 ATCC13032ΔpanB::panB(EC)pECCG117-panD(PS) 1.5 ATCC13032ΔpanB::panB(EC)pECCG117-panD(TC) 5.5

[0136] The results are shown in Table 3 above, confirming that the parent strain, Corynebacterium glutamicum ATCC13032ΔpanB::panB(EC) strain, hardly produces pantothenic acid, and in the strain expressing the aspartate 1-decarboxylase of Corynebacterium glutamicum (ATCC13032ΔpanB::panB(EC)pECCG117-panD(CG)), about 2.0 g / L of pantothenic acid is produced. Among the strains expressing the aspartate 1-decarboxylase derived from an exogenous microorganism, the strain expressing the aspartate 1-decarboxylase derived from Tribolium castaneum ATCC13032ΔpanB::panB(EC)pECCG117-panD(TC) exhibited the highest pantothenic acid production capacity of 5.5 g / L.

[0137] The above results indicate that, among the six enzymes selected in the present invention, the aspartate 1-decarboxylase derived from Tribolium castaneum can not only produce β-alanine, but also can more efficiently produce pantothenic acid.

[0138] According to the above description, it will be understood by those skilled in the art that the present invention can be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the above embodiments should be understood as illustrative and non-restrictive in all aspects. The scope of the present invention should be interpreted as including all changes or modifications from the meaning and scope of the claims described below, and its equivalent concepts are included in the scope of the present invention.

[0139] [Accession number]

[0140] Name of depository: Korea Collection of Microorganisms Accession number: KCCM13076P

[0141] Date of deposit: November 23, 2021

[0146]

Claims

1. A Corynebacterium sp. microorganism producing β-alanine or a β-alanine-derived compound, into which an aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the aspartate 1-decarboxylase is introduced.

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

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

4. The microorganism according to claim 1, in, 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, in, The Corynebacterium microorganism is Corynebacterium glutamicum.

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

7. A method for producing β-alanine or a β-alanine-derived compound, comprising culturing a microorganism of the genus Corynebacterium in a culture medium, into which an aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the aspartate 1-decarboxylase is introduced.

8. The method according to claim 7, further comprising recovering β-alanine or a β-alanine derivative compound from the culture medium or the microorganism according to the culturing.

9. A composition for producing β-alanine or a β-alanine-derived compound, comprising a microorganism of the genus Corynebacterium into which an aspartate 1-decarboxylase derived from Tribolium castaneum or a polynucleotide encoding the aspartate 1-decarboxylase is introduced.

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