Mutant of aspartate 1-decarboxylase from Tribolium castaneum and microorganisms containing the same

Abstract

The present application provides a mutant polypeptide having aspartate 1-decarboxylase activity, a microorganism containing the same, a composition for producing beta-alanine or a beta-alanine-derived compound containing the microorganism, and a method for producing beta-alanine or a beta-alanine-derived compound comprising culturing the microorganism, and has excellent production ability of beta-alanine or a beta-alanine-derived compound.

Description

[Technical field]

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0034233 dated March 18, 2022, and all contents disclosed in the documents of the Korean Patent Application are incorporated herein by reference.

[0002] Provided are a mutant polypeptide of aspartate 1-decarboxylase derived from Tribolium castaneum, a microorganism containing the same, 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 comprising the step of culturing the microorganism. [Background technology]

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

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

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

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

[0007] The present application provides a mutant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to residue 139 in the amino acid sequence of SEQ ID NO:51 has been substituted with another amino acid.

[0008] The present application provides polynucleotides encoding the mutant polypeptides.

[0009] The present application provides a recombinant vector comprising the polynucleotide.

[0010] The present application provides a microorganism comprising the mutant polypeptide and / or a polynucleotide encoding same.

[0011] The present application provides a composition for producing beta-alanine and / or beta-alanine derived compounds, comprising a microorganism containing the mutant polypeptide and / or a polynucleotide encoding the same.

[0012] The present application provides a method for producing beta-alanine and / or beta-alanine derived compounds comprising the step of culturing a microorganism containing said mutant polypeptide and / or a polynucleotide encoding same.

[0013] The present application provides the use of said microorganisms for the production of beta-alanine and / or beta-alanine derived compounds.

[0014] The present application provides the use of said microorganisms for the manufacture of compositions for the production of beta-alanine and / or beta-alanine derived compounds. [Means for solving the problem]

[0015] The present application aims to provide a mutant polypeptide having aspartate 1-decarboxylase (PanD protein) activity that can improve beta-alanine and / or beta-alanine-derived compound production ability, or a recombinant microorganism (strain) having excellent beta-alanine and / or beta-alanine-derived compound production ability into which the mutant polypeptide has been introduced.

[0016] To achieve the above object, one aspect of the present application provides a mutant polypeptide having aspartate 1-decarboxylase activity, which may include a substitution of an amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 with another amino acid.

[0017] Another example provides a polynucleotide encoding (or coding) the polypeptide.

[0018] Another example provides a recombinant vector comprising the polynucleotide, which can be used as an expression vector for the polypeptide.

[0019] Another example provides a microorganism comprising one or more selected from the group consisting of the polypeptide, a polynucleotide encoding the polypeptide, and a recombinant vector comprising the polynucleotide. The microorganism may be a microorganism that produces beta-alanine and / or a beta-alanine derived compound.

[0020] Another embodiment provides a composition for producing beta-alanine and / or beta-alanine derived compounds comprising said microorganism.

[0021] Another example provides a method for producing beta-alanine and / or beta-alanine derived compounds comprising culturing said microorganism.

[0022] This will be explained in more detail below.

[0023] Polypeptides One aspect of the present application provides a mutant polypeptide having an activity of aspartate 1-decarboxylase (or PanD protein). The mutant polypeptide having aspartate 1-decarboxylase activity may be a mutant polypeptide in which a mutation is introduced into the aspartate 1-decarboxylase protein by substitution, deletion, or insertion of one or more amino acid residues.

[0024] The protein to be mutagenized in the present application may be a protein having aspartate 1-decarboxylase activity. The sequence of the protein can be obtained from the public database NCBI, and specifically, the protein may be composed of the amino acid sequence of SEQ ID NO: 51 or may contain the amino acid sequence of SEQ ID NO: 51 and have aspartate 1-decarboxylase activity, but is not limited thereto. That is, without excluding meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO: 51, naturally occurring mutations, or silent mutations thereof, any protein having the same or corresponding activity as a protein containing the amino acid sequence of SEQ ID NO: 51 may be a protein to be mutagenized in the present application. For example, the protein to be mutagenized in the present application may be a protein composed of the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. In addition, proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added are also included within the scope of proteins to be mutated in the present application, so long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to that of the above-mentioned protein.

[0025] In one example, the mutant polypeptide having aspartate 1-decarboxylase activity has an amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51, which is substituted with another amino acid, i.e., tryptophan (Trp, W), histidine (His, H), tyrosine (Tyr, T), alanine (Ala, A), cysteine ​​(Cys, C), proline (Pro, P), serine (Ser, S), leucine (Leu, L), isoleucine (Il, L), or methyl 2-amino-3-propanediol (M2, 2H ... The amino acid may be selected from the group consisting of: arginine (Arg, R), lysine (Lys, K), valine (Val, V), methionine (Met, M), aspartic acid (Asp, D), glutamic acid (Glu, E), glycine (Gly, G), asparagine (Asn, N), glutamine (Gln, Q), and tyrosine (Tyr, Y), and may be substituted with an amino acid different from the original amino acid (phenylalanine, Phe, F). In one embodiment, the mutant polypeptide having aspartate 1-decarboxylase activity may be one in which the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 is replaced with a basic amino acid (arginine, histidine, or lysine), an acidic amino acid (aspartic acid or glutamic acid), a polar amino acid (serine, threonine, asparagine, or glutamine), alanine, valine, isoleucine, leucine, tyrosine, or glycine. In one embodiment, the mutant polypeptide having aspartate 1-decarboxylase activity may be one in which the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 is replaced with threonine, alanine, serine, leucine, isoleucine, valine, aspartic acid, glycine, histidine, or tyrosine. In one embodiment, the mutant polypeptide having aspartate 1-decarboxylase activity may be one in which the amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 is replaced with tyrosine. In one specific example, the aspartate 1-decarboxylase mutant may consist of any one of the amino acid sequences selected from SEQ ID NO:52 to SEQ ID NO:61, or may include any one of the amino acid sequences selected from SEQ ID NO:52 to SEQ ID NO:61.

[0026] The mutant polypeptides of the present application can have properties that result in increased ability to produce beta-alanine and / or beta-alanine derived compounds compared to a wild-type polypeptide that has aspartate 1-decarboxylase activity.

[0027] It is obvious that the mutant polypeptide of the present application includes a mutant polypeptide in which a portion of the amino acid sequence, excluding the amino acid corresponding to the 139th amino acid residue in the amino acid sequence of SEQ ID NO:51, is deleted, modified, substituted, or added, as long as it exhibits aspartate 1-decarboxylase activity.

[0028] For example, the amino acid sequence may have sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally that do not change the function of the mutant polypeptide of the present application.

[0029] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0030] That is, the mutant polypeptide of the present application has an amino acid corresponding to the 139th residue of the amino acid sequence of SEQ ID NO:51 substituted with another amino acid, and has a homology or identity of 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 to the amino acid sequence of SEQ ID NO:51. or may consist of or essentially consist of an amino acid sequence that has, but is not limited to, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.3%, at least 99.5%, at least 99.7%, at least 99.9% to the amino acid sequence of SEQ ID NO:51. In one example, the mutant polypeptide of the present application may have or comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity to the sequence of SEQ ID NO: 51 in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO: 51 is replaced with another amino acid, or may consist of or essentially of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity to the sequence of SEQ ID NO: 51 in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO: 51 is replaced with another amino acid, but is not limited thereto.

[0031] In one example, the variant polypeptide has an amino acid sequence similar to that of SEQ ID NO:51 but differs from that of SEQ ID NO:52 by 70% or more and less than 100%, 75% or more and less than 100%, 80% or more and less than 100%, 85% or more and less than 100%, 90% or more and less than 100%, 95% or more and less than 100%, 96% or more and less than 100%, 97% or more and less than 100%, 98% or more and less than 100%, 98.2% or more and less than 100%, 98.4% or more and less than 100%, 98.6% or more and less than 100%, %, or may comprise, consist of, or consist essentially of, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.3%, at least 99.5%, at least 99.7%, or at least 99.9% and less than 100% sequence homology or identity to the sequence of the present invention, but is not limited to such.

[0032] In the present application, the term "mutated polypeptide" refers to a polypeptide that differs from the amino acid sequence of the mutant polypeptide by conservative substitution and / or modification of one or more amino acids, but maintains its functions or properties. Such mutant polypeptides are generally identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the mutant polypeptide is increased, unchanged, or decreased compared to the mutant polypeptide. In addition, some mutant polypeptides can include mutant polypeptides in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other mutant polypeptides can include mutant polypeptides in which portions have been removed from the N- and / or C-terminus of the mature protein. The term "mutated polypeptide" may be interchangeably used with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant, variant, etc. (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited thereto as long as it is a term used to mean mutated. For the purposes of this application, the mutant polypeptide may be a polypeptide comprising an amino acid sequence in which the amino acid corresponding to the 139th residue from the N-terminus in the amino acid sequence of SEQ ID NO:51 is replaced with another amino acid.

[0033] The mutant polypeptide may also include deletions or additions of amino acids that have minimal effects on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation may be conjugated to the N-terminus of the mutant polypeptide. The mutant polypeptide may also be conjugated to other sequences or linkers so that it can be identified, purified, or synthesized.

[0034] Polynucleotides Another aspect provides a polynucleotide encoding the mutant polypeptide.

[0035] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain of a certain length or more.

[0036] The polynucleotide of the present application may code for a mutant polypeptide in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 is replaced with another amino acid. Alternatively, the polynucleotide of the present application may comprise a nucleotide sequence encoding the mutant polypeptide in which the codons corresponding to the 415th to 417th residues in the nucleotide sequence of SEQ ID NO:62 are replaced with codons encoding other amino acids. As an example of the present application, the polynucleotide of the present application may comprise a nucleotide sequence encoding the amino acid sequence described in any one of SEQ ID NOs:52 to 61. As an example of the present application, the polynucleotide of the present application may have or comprise any one of SEQ ID NOs:63 to 72. In addition, the polynucleotide of the present application may consist of or essentially consist of any one of SEQ ID NOs:63 to 72.

[0037] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the mutant polypeptide of the present application, taking into consideration the degeneracy of codons or the codons preferred in an organism in which the mutant polypeptide of the present application is to be expressed. Specifically, the polynucleotide of the present application has a base sequence having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to any one sequence selected from SEQ ID NO:63 to SEQ ID NO:72. or may consist of or essentially consist of a base sequence having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more to any one of the sequences selected from SEQ ID NO:63 to SEQ ID NO:72, but is not limited thereto.

[0038] In one example, the codon encoding the amino acid corresponding to the 139th position in SEQ ID NO:51 in the sequence having homology or identity may be one of the codons encoding tryptophan, histidine, tyrosine, alanine, cysteine, proline, serine, leucine, isoleucine, arginine, lysine, valine, methionine, aspartic acid, glutamic acid, glycine, asparagine, glutamine, or tyrosine. In one specific example, the codon encoding the amino acid corresponding to the 139th position in SEQ ID NO:51 in the sequence having homology or identity may be one of the codons encoding a basic amino acid (arginine, histidine, or lysine), an acidic amino acid (aspartic acid or glutamic acid), a polar amino acid (serine, threonine, asparagine, or glutamine), alanine, valine, isoleucine, leucine, tyrosine, or glycine. In one embodiment, the codon encoding the amino acid corresponding to the 139th position in SEQ ID NO:51 in the homologous or identical sequence may be one of the codons encoding threonine, alanine, serine, leucine, isoleucine, valine, aspartic acid, glycine, histidine, or tyrosine. In one embodiment, the codon encoding the amino acid corresponding to the 139th position in SEQ ID NO:51 in the homologous or identical sequence may be one of the codons encoding tyrosine.

[0039] Furthermore, the polynucleotide of the present application may include, without limitation, a sequence that can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to the entire or partial polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides having high homology or identity, or polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, hybridize with each other, and polynucleotides having a lower homology or identity than that do not hybridize with each other, or conditions under which polynucleotides having a homology or identity lower ... such as conditions for conventional Southern hybridization (southern Examples of conditions include washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for ELISA hybridization (ELISA), which are 60°C, 1x SSC, 0.1% SDS, specifically 60°C, 0.1x SSC, 0.1% SDS, more specifically 68°C, 0.1x SSC, 0.1% SDS.

[0040] Hybridization requires that two nucleic acids have complementary sequences, even if the stringency of the hybridization allows for mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to an entire sequence, as well as substantially similar nucleic acid sequences.

[0041] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0042] The appropriate stringency for hybridizing said polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0043] As used herein, a polynucleotide (used interchangeably with "gene") or a polypeptide (used interchangeably with "protein") "comprises, consists of, or is expressed as a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including "substantially equivalent sequences" in which meaningless mutations (deletions, substitutions, modifications, and / or additions) have been added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or as not excluding the meaningless mutations).

[0044] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0045] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to the entire sequence or a portion thereof under moderate or high stringent conditions. It is understood that hybridization also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0046] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, e.g., Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, the Needleman-Wunsch algorithm (Needlemanand Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used to determine the sequence, as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later versions) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]:403(1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Database Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity, or identity.

[0047] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48:443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program can be defined as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0048] As used herein, the term "corresponding to" refers to the amino acid residue at a recited position in a polypeptide, or an amino acid residue that is similar or identical or homologous to a recited residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining the particular amino acid of a sequence that references a particular sequence. As used in this application, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0049] For example, any amino acid sequence can be aligned with SEQ ID NO: 52, based on which each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 52. For example, sequence alignment algorithms such as those described herein can identify amino acid positions or positions at which modifications, such as substitutions, insertions, or deletions, occur relative to a query sequence (also referred to as a "reference sequence").

[0050] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16:276-277), etc. can be used, but are not limited thereto, and any sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art can be appropriately used.

[0051] As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to its endogenous activity. The term "enhancement" can be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase can all include exhibiting an activity that was not originally possessed, or exhibiting an activity that is improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before transformation, when the trait is changed by genetic mutation due to natural or artificial factors. This term can be used interchangeably with "activity before modification." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before transformation.

[0052] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity level, expression level, or amount of a product secreted from the polypeptide.

[0053] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the unmodified microorganism. Specifically, the activity of the polypeptide can be enhanced by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0054] Specifically, the enhancement of the polypeptide of the present application is 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome that encodes the polypeptide with a sequence with strong activity; 3) modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene to encode a modified polypeptide so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0055] More specifically, The 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the increase may be achieved by introducing one or more copies of a polynucleotide encoding the polypeptide into a chromosome in a host cell. The introduction into a chromosome may be achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0056] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence having a stronger activity may be, for example, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or a replacement with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

[0057] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent US7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US10584338B2), O2 promoter (US Patent US10273491B2), tkt promoter, and yccA promoter.

[0058] The modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, a replacement with a base sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0059] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or a replacement with an improved amino acid sequence or polynucleotide sequence having a stronger activity or an improved amino acid sequence or polynucleotide sequence having an increased activity. The replacement may be specifically, but is not limited to, by inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may additionally include a selection marker for checking whether or not the chromosome is inserted. The selection marker is as described above.

[0060] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell to produce a polypeptide and increase its activity.

[0061] The codon optimization of the polynucleotide encoding the polypeptide (7) may be codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription or translation in a host cell.

[0062] The step 8) of analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, determining candidate template proteins based on the degree of sequence similarity by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of base proteins is stored, confirming the structure based on the sequence similarity, and selecting and modifying exposed sites to be modified or chemically modified.

[0063] Such enhanced polypeptide activity may refer to, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide expressed relative to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0064] Recombinant Vectors Another example provides a recombinant vector comprising the polynucleotide. The recombinant vector can be used as an expression vector for the polypeptide. The recombinant vector can be for inserting the polynucleotide into the genome of a host cell or for replacing the corresponding gene in the genome of a host cell.

[0065] The vector of the present application can comprise a DNA construct comprising a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of said polypeptide of interest in a suitable host. The expression control region can comprise a promoter capable of initiating transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or can be integrated into the genome itself.

[0066] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pTop33ori vectors can be used.

[0067] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for intracellular chromosome insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of the chromosome insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to check the presence or absence of the insertion of the target nucleic acid molecule, and can be a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

[0068] In the present application, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be any polynucleotide that is inserted into the chromosome of the host cell or extrachromosomally located, as long as it is expressed in the host cell. The polynucleotide may include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure that contains all elements necessary for the polynucleotide to be expressed. The expression cassette may generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0069] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0070] microorganisms Another aspect provides a microorganism comprising one or more (e.g., one or more, two or more, or one, two or three) selected from the group consisting of the mutant polypeptide, a polynucleotide encoding (or coding) the polypeptide, and a recombinant vector comprising the polynucleotide. In one example, the microorganism may be a microorganism having increased (or improved) ability to produce beta-alanine and / or beta-alanine derived compounds.

[0071] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and may include microorganisms in which a specific mechanism has been weakened or enhanced, for example, by the insertion of an exogenous gene and / or the enhancement or inactivation of an endogenous gene, and may also include microorganisms that contain genetic modifications for the production of a desired polypeptide, protein, or product (e.g., aspartate 1-decarboxylase).

[0072] The microorganism of the present application may be, but is not limited to, a microorganism genetically modified through a vector containing a polynucleotide encoding or containing the mutant polypeptide (e.g., a recombinant microorganism). The vector is as described above.

[0073] The microorganism (or strain, recombinant cell) of the present application may be a microorganism having the ability to produce beta-alanine and / or beta-alanine derived compounds or having an improved ability (or production amount) to produce beta-alanine and / or beta-alanine derived compounds.

[0074] The microorganism of the present application may be, but is not limited to, a microorganism naturally having the ability to produce beta-alanine and / or beta-alanine-derived compounds, or a microorganism to which the ability to produce beta-alanine and / or beta-alanine-derived compounds is imparted or improved compared to a microorganism that does not have the ability to produce beta-alanine and / or beta-alanine-derived compounds. The microorganism to which the ability to produce beta-alanine and / or beta-alanine-derived compounds is imparted or improved may be a microorganism to which a mutant polypeptide having the activity of aspartate 1-decarboxylase has been introduced.

[0075] The microorganism (or strain, recombinant cell) has an increased (or improved) ability to produce beta-alanine and / or a beta-alanine-derived compound (or production amount). Having the ability to produce beta-alanine and / or a beta-alanine-derived compound may mean that the microorganism (or strain, recombinant cell) has an improved ability to produce beta-alanine and / or a beta-alanine-derived compound compared to an unmodified microorganism, a cell before recombination, a parent strain, a wild-type strain, or a microorganism into which aspartate 1-decarboxylase derived from another microorganism (e.g., wild-type Tribolium castaneum) has been introduced; or that the microorganism (or strain, recombinant cell) has been imparted with an ability to produce beta-alanine and / or a beta-alanine-derived compound, unlike an unmodified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain that does not have the ability to produce beta-alanine and / or a beta-alanine-derived compound.

[0076] The microorganism of the present application may be, but is not limited to, a microorganism comprising any one or more of a variant polypeptide of the present application, a polynucleotide encoding a variant polypeptide of the present application, and a vector comprising a polynucleotide of the present application; a microorganism modified to express a variant polypeptide of the present application or a polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses a variant polypeptide of the present application or a polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has activity of a variant polypeptide of the present application.

[0077] In one example, the microorganism of the present application may be a Corynebacterium sp. microorganism. The microorganism of the genus Corynebacterium is, for example, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be one or more selected from the group consisting of Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium flavescens.

[0078] The microorganism may have improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds compared to an unmodified microorganism of the same species. In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, but may refer to a wild-type strain or a naturally occurring strain itself, or a strain before its characteristics are changed due to genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain before the mutant polypeptide or polynucleotide encoding the mutant polypeptide of the present application is introduced or is introduced. Alternatively, the "unmodified microorganism" may refer to a strain into which aspartate 1-decarboxylase derived from wild-type Tribolium castaneum or a polynucleotide encoding the same is introduced. The "unmodified microorganism" may be used interchangeably with "pre-modification strain", "pre-modification microorganism", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism". In one example, the unmodified microorganism, which is the subject strain for comparing the increase in the ability to produce beta-alanine and / or beta-alanine-derived compounds, may specifically be a wild-type Corynebacterium glutamicum ATCC13032 strain or a strain in which wild-type Tribolium castaneum panD has been introduced in place of panD of the wild-type Corynebacterium glutamicum ATCC13032 strain, but is not limited thereto.

[0079] The microorganism (or strain, recombinant cell) may further include a mutation that increases the production of beta-alanine and / or beta-alanine derived compounds, and the position of the mutation and / or the type of gene and / or protein to be mutated may be any that increases the production of beta-alanine and / or beta-alanine derived compounds. The recombinant cell may be any cell that can be transformed.

[0080] In one embodiment, the microorganism may have the deposit number KCCM13077P.

[0081] The beta-alanine derived compound may be one or more selected from the group consisting of β-nitropropanoate, β-amino-propionitrile, N-acetyl-β-alanine, L-aspartate, anserine, spermine, carnosine, β-alanyl arginine, quinolinate, pantothenate (or pantothenic acid), malonate semialdyhyde, malonate, acetylene-monocarboxylate, and the like, but is not limited thereto.

[0082] In other examples, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) may have an ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) increased by about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, or about 150% or more compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which aspartate 1-decarboxylase derived from wild-type Tribolium castaneum has been introduced, but is not limited thereto.

[0083] In other examples, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) may have an ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) that is increased by about 2.0 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, or about 2.5 times or more compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which aspartate 1-decarboxylase derived from wild-type Tribolium castaneum has been introduced, but is not limited thereto.

[0084] In other examples, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) may have an increased ability to produce beta-alanine and / or beta-alanine-derived compounds (or production amount) of about 1.0 g / L or more, about 1.1 g / L or more, about 1.2 g / L or more, about 1.3 g / L or more, about 1.4 g / L or more, or about 1.5 g / L or more compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which aspartate 1-decarboxylase derived from wild-type Tribolium castaneum has been introduced, but is not limited thereto.

[0085] More specifically, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or a beta-alanine-derived compound has a higher production capacity (or production amount) than a pre-mutation parent strain, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type red flour beetle has been introduced. may have, but is not limited to, an increase in beta-alanine derived compound production capacity (or production amount) of about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% (or about 2.0-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, or about 2.5-fold; or about 1.0 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L, or about 1.5 g / L).

[0086] In another example, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine derived compounds (or production amount) is characterized by a higher production capacity of beta-alanine and / or beta-alanine derived compounds (e.g., bread) than the parent strain before mutation, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum has been introduced. The present invention is not limited to an increase in the ability (or amount) of (leucine) production by the enzyme, such as erythritol, to produce isopropyl betaine, but may be increased by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 110% or more, about 120% or more, about 125% or more, about 130% or more, about 140% or more, or about 150% or more.

[0087] In another example, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or beta-alanine derived compounds (or production amount) has an improved ability to produce beta-alanine and / or beta-alanine derived compounds (or production amount) compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum has been introduced. may be increased by about 1.05-fold or more, about 1.1-fold or more, about 1.15-fold or more, about 1.2-fold or more, about 1.25-fold or more, about 1.3-fold or more, about 1.35-fold or more, about 1.4-fold or more, about 1.45-fold or more, about 1.5-fold or more, about 1.55-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2-fold or more, about 2.1-fold or more, about 2.2-fold or more, about 2.25-fold or more, about 2.3-fold or more, about 2.4-fold or more, or about 2.5-fold or more, but is not limited thereto.

[0088] In another example, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or a beta-alanine-derived compound (or production amount) has an ability to produce beta-alanine and / or a beta-alanine-derived compound (or production amount) of about 0.05 g / L or more, about 0.06 g / L or more, about 0.07 g / L or more, about 0.08 g / L or more, about 0.09 g / L or more, about 0.1 g / L or more, about 0.13 g / L or more, about 0.16 g / L or more, compared to a pre-mutation parent strain, an unmodified microorganism, a microorganism into which an aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which an aspartate 1-decarboxylase derived from a wild-type Tribolium castaneum has been introduced. Above, about 0.19g / L or more, about 0.2g / L or more, about 0.23g / L or more, about 0.26g / L or more, about 029g / L or more, about 0.3g / L or more, about 0.33g / L or more Above, about 0.36g / L or more, about 0.39g / L or more, about 0.4g / L or more, about 0.43g / L or more, about 0.46g / L or more, about 0.49g / L or more, about 0.5g / L or more The increase may be, but is not limited to, about 0.53 g / L or more, about 0.56 g / L or more, about 0.59 g / L or more, about 0.6 g / L or more, about 0.63 g / L or more, about 0.67 g / L or more, about 0.69 g / L or more, about 0.7 g / L or more, about 0.8 g / L or more, about 0.9 g / L or more, or about 1.0 g / L or more.

[0089] More specifically, the microorganism (or strain, recombinant cell) having improved (increased) ability to produce beta-alanine and / or a beta-alanine-derived compound (or production amount) has an ability to produce beta-alanine and / or a beta-alanine-derived compound (or production amount) of about 10% or more compared to the parent strain before mutation, an unmodified microorganism, a microorganism into which aspartate 1-decarboxylase derived from another microorganism has been introduced, or a microorganism into which aspartate 1-decarboxylase derived from wild-type Tribolium castaneum has been introduced. The increase may be, but is not limited to, about 15%, about 20%, about 25%, about 35%, about 40%, about 55%, about 100%, or about 125% (or about 1.1-fold, about 1.15-fold, about 1.2-fold, about 1.25-fold, about 1.35-fold, about 1.4-fold, about 1.55-fold, about 2-fold, or 2.25-fold; or about 0.06 g / L, about 0.1 g / L, about 0.13 g / L, about 0.16 g / L, about 0.23 g / L, about 0.26 g / L, about 0.36 g / L, about 0.5 g / L, or about 0.6 g / L).

[0090] The term "about" includes, but is not limited to, all numerical values ​​in a range equal to or similar to the numerical value following the term about, including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc.

[0091] Another aspect is to provide a composition for producing beta-alanine and / or beta-alanine derived compounds comprising a microorganism of the present application, a medium in which said microorganism is cultured, or a combination thereof.

[0092] The compositions of the present application may additionally contain any suitable excipients commonly used in compositions for the production of beta-alanine and / or beta-alanine derived compounds, such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0093] In the composition of the present application, the microorganism (strain), medium, etc. are as described in the other embodiments above.

[0094] Another embodiment provides a method for producing beta-alanine and / or beta-alanine derived compounds comprising culturing a microorganism of the present application in a medium.

[0095] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application can include culturing the microorganism of the present application in a medium. The microorganism of the present application is as described above.

[0096] In the present application, "culturing" refers to growing a microorganism containing the variant polypeptide of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be performed using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch type.

[0097] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing the microorganism of the present application as the main ingredient, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular limitation, and the microorganism of the present application can be cultured in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins under aerobic conditions while controlling temperature, pH, etc.

[0098] Specifically, the culture medium for the microorganism of the present application, for example, a Corynebacterium sp. strain, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0099] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residue, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other suitable amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0100] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0101] The phosphorus source may include potassium monophosphate, potassium diphosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may also include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0102] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foam generation may be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain anaerobic and microaerobic states, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0103] In the culture of the present application, the culture temperature can be maintained at 20 to 45° C., specifically 25 to 40° C., and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0104] The beta-alanine and / or beta-alanine derived compounds produced by the cultures of the present application may be secreted into the medium or remain intracellularly.

[0105] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application may additionally include, for example prior to the culturing step, a step of preparing a microorganism (strain) of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order).

[0106] The method for producing beta-alanine and / or beta-alanine derived compounds of the present application may further include a step of recovering beta-alanine and / or beta-alanine derived compounds from the culture medium (culture medium) or microorganism (e.g., Corynebacterium sp. strain). The recovery step may be additionally included after the culture step.

[0107] Said recovery may be to collect the desired beta-alanine and / or beta-alanine derived compounds using a suitable method known in the art, such as a batch, continuous or fed-batch culture method of the microorganism of the present application. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination of these methods can be used, and the desired beta-alanine and / or beta-alanine derived compounds can be collected from the medium or the microorganism using a suitable method known in the art.

[0108] In addition, the method for producing beta-alanine and / or beta-alanine derived compounds of the present application may additionally include a purification step. The purification may be carried out using any suitable method known in the art. In one example, when the method for producing beta-alanine and / or beta-alanine derived compounds of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously in any order, or may be carried out simultaneously or integrated into one step, but is not limited thereto.

[0109] Another embodiment provides a use of the microorganism of the present application for the production of beta-alanine and / or beta-alanine derived compounds or for the manufacture of a composition for the production of beta-alanine and / or beta-alanine derived compounds, wherein the microorganism of the present application, the beta-alanine and / or beta-alanine derived compounds or the composition for the production of beta-alanine and / or beta-alanine derived compounds are as described above. Effect of the Invention

[0110] The present application provides a mutant polypeptide having aspartate 1-decarboxylase activity, a microorganism containing the same, 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 comprising a step of culturing the microorganism, wherein the microorganism has excellent ability to produce beta-alanine and / or a beta-alanine-derived compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] The present invention will be described in more detail below with reference to the following examples, which are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0112] Example 1. Random mutant strain creation using NTG-based artificial mutagenesis and selection of strain expressing panD mutant polypeptide (mutant) In this example, in order to obtain a microbial mutant with improved beta-alanine production, a mutation was induced in the parent strain Corynebacterium glutamicum ATCC13032 (ATCC13032ΔpanD::panD(TC)) into which the panD gene (the gene encoding aspartate 1-decarboxylase, or PanD protein) derived from Tribolium castaneum was introduced.

[0113] The ATCC13032ΔpanD::panD(TC) strain was constructed as follows. First, a vector was constructed to delete the panD gene endogenously present in the Corynebacterium glutamicum ATCC13032 strain. PCR was performed using primers of SEQ ID NOs: 73 and 74 and SEQ ID NOs: 75 and 76 with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. PCR was performed under the following conditions: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 30 times. As a result, gene fragments of 1000 bp upstream of the panD gene and 1000 bp downstream of the panD gene were obtained, and each amplified product was purified using QIAGEN's PCR Purification kit and used as the insert DNA fragment for vector construction.

[0114] The pDCM2 (Korea Patent No. 2278000) vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the above DNA fragments (a 1000 bp gene fragment from the upstream region of the panD gene and a 1000 bp gene fragment from the downstream region of the panD gene) were cloned at a molar concentration (M) of 2:1:1 using the Infusion Cloning Kit from Takara according to the manual provided, to prepare a vector pDCM2_ΔpanD for deleting the panD gene on the chromosome.

[0115] To prepare the panD gene from Tribolium castaneum, PCR was performed using primers 77 and 78 with genomic DNA extracted from Tribolium castaneum as a template. PCR was performed by repeating denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and polymerization at 72℃ for 1 minute and 30 seconds 30 times, resulting in a 1640 bp DNA fragment. To obtain the lysC promoter from Corynebacterium glutamicum, PCR was performed in the same manner as in the previous example using primers 79 and 80 with Corynebacterium glutamicum genomic DNA as a template to obtain a promoter DNA fragment. The pDCM2_ΔpanD vector was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The DNA fragment obtained was then cloned using the Infusion Cloning Kit (TaKaRa) according to the instructions provided, at a molar concentration (M) of 2:1:1, to produce the vector pDCM2_ΔpanD::panD(TC) for introducing the panD gene derived from Tribolium castaneum into the chromosome.

[0116] The constructed vector pDCM2_ΔpanD::panD(TC) was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain (ΔpanD::panD(TC)) in which E. coli-derived panD was introduced on the chromosome was selected through a second crossover process. The presence or absence of appropriate substitution of E. coli-derived panD was confirmed using the MASA (Mutant Allele Specific Amplification) PCR technique (Takeda et al., Hum. Mutation, 2, 112-117(1993)) with the following primer combinations. That is, the primary determination was made by selecting strains that were amplified with the primer combinations (SEQ ID NO: 81 and 80 and SEQ ID NO: 77 and 82) corresponding to Tribolium castaneum panD, and the panD sequence of the selected strains was analyzed using the primer combinations of SEQ ID NO: 81 and SEQ ID NO: 82 to complete the secondary selection.

[0117] The selected Corynebacterium glutamicum ATCC13032 ΔpanD::panD (TC) strain was activated by culturing in activation medium for 16 hours, inoculated into a minimal medium sterilized at 121°C for 15 minutes, and cultured for 14 hours, after which 5 mL of culture was harvested. The harvested culture was washed with 100 mM citric buffer, and then NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) was added to a final concentration of 200 mg / L, treated for 20 minutes, and then washed with 100 mM phosphate buffer. The NTG-treated strain was smeared on a minimal medium to measure the mortality, which was shown to be 85%. The surviving cells were inoculated into a production medium and cultured, and finally, the mutant strain showing the best beta-alanine production ability was selected and named Corynebacterium glutamicum CJVB5-03-1.

[0118] The composition of the medium used in this example is as follows.

[0119] <Activation medium> Beef extract 1%, polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2

[0120] <Production medium> Glucose 10%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 6.7 mg / L, biotin 50 μg / L, thiamine·HCl 100 μg / L, pH 7.2

[0121] <Minimal medium> Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate monobasic 0.1%, urea 0.1%, thiamine 0.001%, biotin 200μg / L, agar 2%, pH 7.2

[0122] To compare the beta-alanine production ability of Corynebacterium glutamicum ΔpanD::panD(TC) and the mutant strain Corynebacterium glutamicum CJVB5-03-1 obtained above, the Corynebacterium glutamicum ΔpanD::panD(TC) strain and the mutant strain CJVB5-04 were inoculated into 250-ml corner baffle flasks containing 25 ml of production medium, respectively, and then cultured at 33°C for 48 hours with shaking at 200 rpm.

[0123] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and the supernatant was diluted 1 / 10 with TDW (triple distilled water) and then subjected to HPLC analysis to measure the concentration of beta-alanine. The results are shown in Table 1 below.

[0124] [Table 1]

[0125] As a result, as shown in Table 1, Corynebacterium glutamicum ATCC13032 ΔpanD::panD(TC) produced beta-alanine at a concentration of 0.5 g / L, while the mutant Corynebacterium glutamicum CJVB5-03-1 according to the present application produced beta-alanine at a concentration of 1.2 g / L, demonstrating that beta-alanine productivity was increased by about 2.4 times (about 140% increase) compared to the parent strain.

[0126] Based on the above results, genome sequencing of genes in the biosynthetic pathway that synthesizes beta-alanine from pyruvate was performed, and random mutations were confirmed in the panD gene derived from Tribolium castaneum in the mutant Corynebacterium glutamicum CJVB5-03-1.

[0127] Specifically, it was confirmed that the sequence encoding phenylalanine (F), the 139th amino acid of the wild-type PanD protein of Tribolium castaneum, was mutated to a sequence encoding tyrosine (Y). The amino acid sequence of the mutant PanD protein (F139Y, in which F (phenylalanine), the 139th amino acid of wild-type PanD (SEQ ID NO: 51) derived from Tribolium castaneum, is mutated to Y (tyrosine)) is shown in SEQ ID NO: 52.

[0128] From the above results, it was confirmed that the mutant strain obtained through random mutation method was capable of producing beta-alanine with high efficiency and high yield.

[0129] Example 2. Confirmation of pantothenic acid production ability of aspartate 1-decarboxylase mutant polypeptide It was confirmed that the beta-alanine producing ability of the mutant polypeptide of aspartate 1-decarboxylase of Tribolium castaneum was increased in Example 1. In addition, in order to confirm the pantothenic acid producing ability of the mutant polypeptide having aspartate 1-decarboxylase activity of Tribolium castaneum, a microorganism with enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase (PanB) was produced.

[0130] First, a vector was constructed to delete the endogenous panB gene (the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase, or PanB protein) present in Corynebacterium glutamicum ATCC13032. PCR was performed using primers of SEQ ID NOs: 41 and 42 and SEQ ID NOs: 43 and 44 with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. PCR was performed under the following conditions: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, repeated 30 times. As a result, gene fragments of 1,000 bp upstream of the panB gene and 1,000 bp downstream of the panB gene were obtained, and each amplified product was purified using QIAGEN's PCR Purification kit and used as the insert DNA fragment for vector construction.

[0131] The pDCM2 (Korea Patent No. 2278000) vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the DNA fragments (a 1,000 bp gene fragment from the upstream region of the panB gene and a 1,000 bp gene fragment from the downstream region of the panB gene) were cloned at a molar concentration (M) of 2:1:1 using the Infusion Cloning Kit from Takara according to the manual provided, to produce the vector pDMC2_ΔpanB for deleting the panB gene on the chromosome.

[0132] To prepare the panB gene from E. coli, PCR was performed using primers 47 and 48 with the genomic DNA of E. coli K12 wild-type strain (KCTC1116) as a template. PCR was performed by repeating denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute 30 times, resulting in a 795 bp DNA fragment. To obtain the lysC promoter from Corynebacterium glutamicum, PCR was performed in the same manner as in the previous example using primers 45 and 46 with Corynebacterium glutamicum genomic DNA as a template to obtain a promoter DNA fragment. The pDMC2_ΔpanB vector was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The DNA fragment obtained was then cloned using the Infusion Cloning Kit from Takara Corporation according to the instructions provided, at a molar concentration (M) of 2:1:1, to produce the vector pDMC2_ΔpanB::panB(EC) for introducing the panB gene derived from E. coli into the chromosome.

[0133] The constructed vector pDMC2_ΔpanB::panB(EC) was transformed into Corynebacterium glutamicum ATCC13032 ΔpanD::panD(TC) and CJVB5-03-1 by electroporation, and the strains (ΔpanB::panB(EC)) in which the E. coli-derived panB gene was introduced into the chromosome were obtained through a second crossover process. The appropriate substitution of E. coli-derived panB was confirmed using the MASA (Mutant Allele Specific Amplification) PCR technique (Takeda et al., Hum.Mutation, 2, 112-117(1993)) with the following primer combinations. That is, the strains in which the E. coli panB gene was amplified with the primer combinations (sequences 49 and 46 and sequences 47 and 50) were selected as the primary determination, and the panB gene sequences of the selected strains were secondarily confirmed by analyzing the primer combinations of sequence numbers 49 and 50.

[0134] In order to confirm the productivity of pantothenic acid, the prepared strains were inoculated into 250 ml corner baffle flasks containing 25 ml of production medium having the composition as described in the above Examples, and cultured at 33° C. for 48 hours with shaking at 200 rpm to produce pantothenic acid. The results are shown in Table 2 below.

[0135] [Table 2]

[0136] As a result, as shown in Table 2, Corynebacterium glutamicum ATCC13032 ΔpanD::panD(TC) ΔpanB::panB(EC) produced pantothenic acid at a concentration of 0.4 g / L, while Corynebacterium glutamicum CJVB5-04 ΔpanB::panB(EC) of the present application produced pantothenic acid at a concentration of 0.9 g / L, demonstrating that the productivity of pantothenic acid was increased by about 2.25 times (about 125% increase) compared to the parent strain.

[0137] The above results mean that the aspartate 1-decarboxylase mutant polypeptide derived from Tribolium castaneum in the present application can more efficiently produce not only beta-alanine but also pantothenic acid.

[0138] Example 3. Construction of expression vector for mutant PanD with aspartate 1-decarboxylase activity As confirmed in Example 1, in order to confirm that the mutation at the position corresponding to the 139th position of the PanD protein (wild type, SEQ ID NO: 51) derived from Tribolium castaneum is important for increasing beta-alanine production ability, a mutant in which the phenylalanine at the 139th position of the PanD protein (wild type, SEQ ID NO: 51) derived from Tribolium castaneum was replaced with another amino acid was prepared and its effect was confirmed.

[0139] Using the wild-type Tribolium castaneum panD gene as a template, PCR was performed with the primer sequences in Table 3 to amplify a DNA fragment encoding aspartate 1-decarboxylase. The PCR was performed using PfuUltraTM high-fidelity DNA polymerase (Stratagene) 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, a 430 bp DNA fragment from the 5' upstream site and a 1224 bp DNA fragment from the 3' downstream site were obtained, centered on the mutation in the gene encoding aspartate 1-decarboxylase (a mutation in the codon encoding the 139th residue of the PanD protein).

[0140] To obtain the PLM1 promoter from Corynebacterium glutamicum, PCR was performed in the same manner as described above using the genomic DNA of Corynebacterium glutamicum (ATCC13032) as a template and primers of SEQ ID NOs: 39 and 40 to obtain a promoter DNA fragment.

[0141] [Table 3]

[0142] The pECCG117 (Korea Patent Registration No. 10-0057684) vector, which had been treated with the restriction enzyme BamHI and then heat-treated at 65°C for 20 minutes, and the resulting DNA fragments (each panD and PLM1 promoter) were cloned according to the manual provided using the Infusion Cloning Kit from Takara at a molar concentration (M) of 2:1:1:1 (pECCG117 vector: upstream site of panD: downstream site of panD: promoter) to obtain plasmids. The names of the obtained 11 plasmids and the introduced gene information are shown in Table 4.

[0143] [Table 4]

[0144] Example 4. Evaluation of pantothenic acid production ability of mutant PanD The 10 mutant plasmids and pECCG117-panD(WT) prepared in Example 3 were introduced into Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC) strain by electric pulse method, and each transformed strain was obtained by plating on a selection medium containing 25 mg / L kanamycin. Then, flask evaluation was performed in the same manner as in Example 2, and the results are shown in Table 5 below.

[0145] [Table 5]

[0146] As can be seen from Table 5 above, the strain into which the wild-type panD was introduced (ATCC13032ΔpanB::panB(EC)pECCG117-panD(WT)) and the strain into which the mutant panD was introduced all showed increased pantothenic acid production ability.

[0147] In particular, a total of 10 mutant strains (F139T, F139A, F139S, F139L, F139I, F139V, F139D, F139G, F139H, F139Y), including a mutant strain expressing mutant PanD in which the 139th amino acid of the wild-type PanD protein was mutated to tyrosine, were confirmed to produce pantothenic acid at a higher level than strains expressing the wild-type PanD protein, and one of the mutant strains (F139Y) produced more than twice as much pantothenic acid as the wild-type. From these results, it was confirmed that the 139th position of the PanD protein derived from Tribolium castaneum is an important position for activity, and that when this position is substituted with another amino acid, it is an important position that affects the ability to produce pantothenic acid.

[0148] The ATCC13032ΔpanB::panB(EC)pECCG117-panD(F139Y) strain (named Corynebacterium glutamicum CV03-5004), which was confirmed to have the best pantothenic acid production ability in this example, was deposited at the Korea Microorganism Collection, Hongje-dong, Seodaemun-gu, Seoul, Republic of Korea, on November 23, 2021, and was assigned the deposit number KCCM13077P.

[0149] From the above description, it should be understood by those skilled in the art to which the present application pertains that the present invention can be embodied in other specific forms without changing its technical concept or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below and their equivalent concepts rather than the above detailed description. [Accession number]

[0150] Name of depository institution: Korea Microorganism Collection Center Accession number: KCCM13077P Date of acceptance: 20211123 JPEG2025508254000006.jpg214168

Claims

1. A mutant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 has been replaced with another amino acid.

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

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

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

51.

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

6. A microorganism comprising a mutant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 has been replaced with another amino acid, or a polynucleotide encoding the same.

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

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

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

10. A composition for producing beta-alanine or a beta-alanine-derived compound, comprising a microorganism containing a mutant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 is substituted with another amino acid, or a polynucleotide encoding the same.

11. A method for producing beta-alanine or a beta-alanine-derived compound, comprising the step of culturing in a medium a microorganism containing a mutant polypeptide having aspartate 1-decarboxylase activity, in which the amino acid corresponding to the 139th residue in the amino acid sequence of SEQ ID NO:51 has been substituted with another amino acid, or a polynucleotide encoding the same.

12. The method for producing beta-alanine or a beta-alanine derived compound according to claim 11, further comprising the step of recovering beta-alanine or a beta-alanine derived compound from the cultured microorganism, the medium, or both after the culturing step.

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