Microorganisms with enhanced 3-methyl-2-oxobutanoate hydroxymethyltransferase activity and uses thereof

Abstract

Provided are 3-methyl-2-oxobutanoate hydroxymethyltransferase mutants, microorganisms having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, compositions for producing pantothenic acid and / or pantoic acid comprising the microorganisms, and methods for producing pantothenic acid and / or pantoic acid comprising culturing the microorganisms.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0060009, dated May 10, 2021, and all contents disclosed in the documents of said Korean Patent Application are incorporated herein by reference.

[0002] Provided are a 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant, a microorganism with enhanced 3-methyl-2-oxobutanoate hydroxymethyltransferase activity, a composition for producing pantothenic acid and / or pantoic acid containing the microorganism, and a method for producing pantothenic acid and / or pantoic acid comprising culturing the microorganism. [Background technology]

[0003] Pantothenic acid, also known as vitamin B5, is a substance belonging to the vitamin B complex and is one of the commercially important substances with diverse applications in cosmetics, medicines, human nutrition, animal nutrition, etc. Pantothenic acid is a structure in which beta-alanine is linked to pantoic acid via an amide bond.

[0004] Pantothenic acid or pantoic acid can be produced chemically or biotechnologically by fermentation of suitable microorganisms in a suitable medium. The advantage of the biotechnological production method using microorganisms is that the desired stereoisomeric D-form of pantothenic acid or pantoic acid is formed.

[0005] Therefore, there is a need to develop a microorganism that has an advantageous effect in biotechnologically producing pantothenic acid and / or pantoic acid, and a technology for using the microorganism to produce pantothenic acid and / or pantoic acid 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] One example of the present application provides a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase. In one specific example, (1) the polypeptide may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid. In another specific example, (2) the polypeptide may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid and the amino acid corresponding to the 116th residue from the N-terminus is substituted with another amino acid. As described above, counting amino acids from the N-terminus in an amino acid sequence can mean counting methionine (Met, M) translated from the start codon as the first amino acid.

[0008] Another example provides a polynucleotide encoding the polypeptide.

[0009] Another example provides a recombinant vector comprising the polynucleotide. The recombinant vector may be an expression vector.

[0010] Another example provides a microorganism that produces pantothenic acid or pantoic acid, which contains one or more (one, two, or all) selected from the group consisting of the polypeptide, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide, and which has enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

[0011] The microorganism is (1) One or more (one, two, or all) selected from the group consisting of a polypeptide in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide; or (2) It may comprise one or more (one, two, or all) selected from the group consisting of (1) above, a polypeptide in which the amino acid corresponding to the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide.

[0012] The microorganism may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. The microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0013] Another example provides a composition for producing pantothenic acid or pantoic acid, comprising the microorganism.

[0014] Another example provides the use of said microorganisms for the production of pantothenic acid and / or pantoic acid.

[0015] Another example provides a method for producing pantothenic acid or pantoic acid, which includes culturing the microorganism in a medium. The method may further include recovering pantothenic acid or pantoic acid from the cultured microorganism, the medium, or both after the culturing step. [Means for solving the problem]

[0016] In this specification, we aim to search for 3-methyl-2-oxobutanoate hydroxymethyltransferase or a mutant thereof that can improve the ability to produce pantothenic acid and / or pantoic acid, and to provide a recombinant strain with excellent ability to produce pantothenic acid and / or pantoic acid by introducing this into a microorganism.

[0017] In the present specification, it has been confirmed that a microorganism expressing 3-methyl-2-oxobutanoate hydroxymethyltransferase has excellent pantothenic acid productivity, and that the pantothenic acid productivity is further increased when an amino acid substitution mutation is introduced into a specific position of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

[0018] One example provides a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase. In one specific example, the polypeptide may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid. In another specific example, the polypeptide may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid and the amino acid corresponding to the 116th residue from the N-terminus is substituted with another amino acid. As described above, counting amino acids from the N-terminus in an amino acid sequence may mean counting methionine (Met, M) translated from the start codon as the first amino acid.

[0019] Another example provides a polynucleotide encoding the polypeptide.

[0020] Another example provides a recombinant vector containing the polynucleotide, which can be used as an expression vector for the polypeptide.

[0021] Another example provides a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, which may be a microorganism that produces pantothenic acid or pantoic acid.

[0022] The activity of the 3-methyl-2-oxobutanoate hydroxymethyltransferase may be enhanced by mutating an endogenous 3-methyl-2-oxobutanoate hydroxymethyltransferase of the microorganism, or by introducing an exogenous 3-methyl-2-oxobutanoate hydroxymethyltransferase or a mutant thereof.

[0023] In one specific example, the 3-methyl-2-oxobutanoate hydroxymethyltransferase with enhanced activity may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 has been substituted with another amino acid. Alternatively, the 3-methyl-2-oxobutanoate hydroxymethyltransferase with enhanced activity may comprise an amino acid sequence in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 has been substituted with another amino acid and the amino acid corresponding to the 116th residue from the N-terminus has been substituted with another amino acid.

[0024] The microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase is (1) one or more selected from the group consisting of a polypeptide in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide; or (2) It may comprise one or more selected from the group consisting of (1) above, a polypeptide in which the amino acid corresponding to the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide.

[0025] A microorganism in which the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase is enhanced can have a higher ability to produce pantothenic acid and / or pantoic acid than a microorganism of the same species in which 3-methyl-2-oxobutanoate hydroxymethyltransferase is not enhanced (e.g., in which the amino acid corresponding to the 159th residue and / or the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is not substituted with another amino acid).

[0026] Another example provides a composition for producing pantothenic acid and / or pantoic acid, which comprises a microorganism in which the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase is enhanced.

[0027] Another example provides a method for producing pantothenic acid and / or pantoic acid, comprising culturing a microorganism in which the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase is enhanced.

[0028] This will be explained in more detail below.

[0029] Polypeptides In this specification, pantothenic acid (e.g., D-pantothenic acid) is a compound having the structure shown in Chemical Formula 1, and is a vitamin (vitamin B5) in which β-alanine is linked to pantoic acid via an amide bond. It is a component of coenzyme A (CoA) and acyl carrier protein (ACP), and is involved in various metabolic functions of living organisms. [ka]

[0030] Pantoic acid (eg, D-pantoic acid) is a compound having the structure shown in Chemical Formula 2 and is a component of a variety of biologically active compounds. [ka]

[0031] As used herein, 3-methyl-2-oxobutanoate hydroxymethyltransferase is an enzyme that catalyzes the biosynthesis of tetrahydrofolate and 2-dihydropantoate from 5,10-methylenetetrahydrofolate, 3-methyl-2-oxobutanoate, and water.

[0032] In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase may be a mutant that has been mutated by substitution, deletion, or insertion of one or more amino acid residues.

[0033] In one example, the mutant 3-methyl-2-oxobutanoate hydroxymethyltransferase may have the amino acid corresponding to residue 159 in the amino acid sequence of SEQ ID NO: 37 substituted with another amino acid different from the original amino acid, i.e., an amino acid selected from the group consisting of arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), tyrosine (Y), phenylalanine (F), tryptophan (W), and glycine (G). In one specific example, the 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant may have the amino acid corresponding to residue 159 in the amino acid sequence of SEQ ID NO: 37 substituted with another amino acid, i.e., arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), or tyrosine (Y). It is clear that the mutants of the present application include those in which a portion of the amino acid sequence, excluding the amino acid corresponding to amino acid residue 159 in the amino acid sequence of SEQ ID NO: 37, has been deleted, modified, substituted, or added, as long as they exhibit the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

[0034] In one example, the variant can include a polypeptide in which the amino acid corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid in an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 37. In other words, variants of the present application include polypeptides that contain a substitution with another amino acid at the position corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37, have at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% sequence homology or identity to the amino acid sequence of SEQ ID NO: 37, and have 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.

[0035] In one specific example, the 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant may include, but is not limited to, an amino acid sequence selected from SEQ ID NOs: 110 to 125. It is clear that a mutant consisting of any one of SEQ ID NOs: 110 to 125, in which a portion of the amino acid sequence, excluding the amino acid corresponding to residue 159, is deleted, modified, substituted, or added, is included in the mutant of the present application as long as it exhibits 3-methyl-2-oxobutanoate hydroxymethyltransferase activity. In one example, the mutant may include a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to any one of the amino acid sequences selected from SEQ ID NOs: 110 to 125, while retaining the amino acid corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37. In other words, a polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity that contains a substitution with another amino acid at the position corresponding to the 159th residue in the amino acid sequence of SEQ ID NO: 37 and has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% sequence homology or identity with any one of the amino acid sequences selected from SEQ ID NO: 110 to SEQ ID NO: 125 is included in the variants of the present application.

[0036] In one example, the mutant of 3-methyl-2-oxobutanoate hydroxymethyltransferase is (1) the amino acid corresponding to the 159th residue of 3-methyl-2-oxobutanoate hydroxymethyltransferase in the amino acid sequence of SEQ ID NO: 37 is replaced with another amino acid, i.e., an amino acid selected from the group consisting of arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), and tyrosine (Y), which is different from the original amino acid; and (2) The amino acid corresponding to the 116th amino acid residue of 3-methyl-2-oxobutanoate hydroxymethyltransferase in the amino acid sequence of SEQ ID NO: 37 may be substituted with another amino acid different from the original amino acid, that is, an amino acid selected from the group consisting of aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), alanine (A), valine (V), isoleucine (I), leucine (L), and methionine (M).

[0037] It is clear that the above mutants, even if they have a partial deletion, modification, substitution, or addition of an amino acid sequence excluding the amino acid residues corresponding to the 159th and 116th residues in the amino acid sequence of SEQ ID NO: 37, are included in the mutants of the present application as long as they exhibit the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

[0038] In one example, the variant may include a polypeptide in which (1) the amino acid corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37 and (2) the amino acid corresponding to residue 116 of the amino acid sequence of SEQ ID NO: 37 are substituted with other amino acids in an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 37. In other words, a polypeptide that contains a substitution with another amino acid at (1) the position corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37 and (2) the position corresponding to residue 116 of the amino acid sequence of SEQ ID NO: 37, has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity to the amino acid sequence of SEQ ID NO: 37 but less than 100%, and has 3-methyl-2-oxobutanoate hydroxymethyltransferase activity is included in the variants of the present application.

[0039] In one specific example, the 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant may include, but is not limited to, the amino acid sequence of SEQ ID NO: 128. It is clear that a mutant consisting of the amino acid sequence of SEQ ID NO: 128, in which a portion of the amino acid sequence excluding (1) the amino acid corresponding to residue 159 and (2) the amino acid corresponding to residue 116 is deleted, modified, substituted, or added, is included in the mutant of the present application as long as it exhibits the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase. In one example, the variant may comprise a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity to the amino acid sequence of SEQ ID NO: 128, in which (1) the amino acid corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37 and (2) the amino acid corresponding to residue 116 of the amino acid sequence of SEQ ID NO: 37 are fixed. In other words, a polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity that contains a substitution with another amino acid at (1) the position corresponding to residue 159 of the amino acid sequence of SEQ ID NO: 37 and (2) the position corresponding to residue 116 of the amino acid sequence of SEQ ID NO: 37 and has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity, but less than 100%, with the amino acid sequence of SEQ ID NO: 128 is included in the variants of the present application.

[0040] microorganisms In this specification, (1) one or more selected from the group consisting of a polypeptide in which the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide; or (2) The polypeptide (1) and the polypeptide (1) in which the amino acid corresponding to the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide, and a recombinant vector containing the polynucleotide. A microorganism that produces pantothenic acid or pantoic acid is provided.

[0041] As used herein, the term "microorganism with enhanced 3-methyl-2-oxobutanoate hydroxymethyltransferase activity" refers to a microorganism that was previously unable to produce pantothenic acid and / or pantoic acid but has been engineered (mutated) to express the aforementioned polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity, thereby enabling the microorganism to produce pantothenic acid and / or pantoic acid, or to a microorganism that has higher pantothenic acid and / or pantoic acid productivity than its original pantothenic acid and / or pantoic acid productivity. As used herein, the term "microorganism" encompasses unicellular bacteria and may be used interchangeably with "cell." In order to distinguish the microorganism before mutation to express the polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity from the mutated microorganism, the term "parent microorganism or parent strain" or "host cell" is used.

[0042] In one example, the microorganism may be one or more species selected from the group consisting of microorganisms of the genus Corynebacterium, microorganisms of the genus Escherichia, etc. The microorganisms of the genus Corynebacterium may include, but are not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, etc. More specifically, the Corynebacterium microorganism may be Corynebacterium glutamicum, and the Escherichia strain may be Escherichia coli.

[0043] As used herein, a microorganism in which the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase has been enhanced may be one into which a gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase has been introduced.

[0044] As used herein, a "microorganism with enhanced 3-methyl-2-oxobutanoate hydroxymethyltransferase activity" may refer to a microorganism that expresses a polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity, the parent strain of which has been subjected to a mutation (manipulation) that causes the parent strain to express the polypeptide. The microorganism may contain one or more elements selected from the group consisting of a polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity, a polynucleotide encoding the polypeptide, and a recombinant vector comprising the polynucleotide. In one example, the mutation that causes the parent strain to express the polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity may be achieved by introducing the polynucleotide encoding the polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity described above, or a recombinant vector comprising the polynucleotide, into the parent strain. The polynucleotide encoding a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase introduced into the parent strain in this manner may replace or be included additionally in addition to the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase present in the parent strain.

[0045] In one embodiment, the microorganism expressing a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase has accession number KCCM12973P (designated Corynebacterium glutamicum CV03-5002).

[0046] As used herein, when a polynucleotide (which may be used interchangeably with "gene") or a polypeptide (which may be used interchangeably with "protein") "comprises, consists of, or is expressed by a specific nucleic acid sequence or amino acid sequence," this can mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence or amino acid sequence, and is interpreted as including "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and / or additions) have been made 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 such mutations).

[0047] In one example, the nucleic acid sequences or amino acid sequences provided herein may be modified by conventional mutagenesis methods, such as directed evolution and / or site-directed mutagenesis, to the extent that their original or intended function is maintained. In one example, a polynucleotide or polypeptide "comprising or consisting of a specific nucleic acid sequence or amino acid sequence" can mean that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially contains an amino acid sequence that has 60% or more, 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, 99% or more, 99.5% or more, or 99.9% or more homology to the specific nucleic acid sequence or amino acid sequence, and maintains its original and / or intended function. In this specification, the target function may refer to a function that increases or imparts the ability of a microorganism to produce pantothenic acid and / or pantoic acid.

[0048] The nucleic acid sequences described herein are modified in various ways in the coding region, taking into account the codons preferred in the microorganism in which the protein (lysine excretion protein) is to be expressed due to codon degeneracy, without changing the amino acid sequence and / or function of the protein expressed from the coding region.

[0049] As used herein, the term "identity" refers to the degree of identity with a given nucleic acid or amino acid sequence, and is expressed as a percentage (%). In the case of nucleic acid sequences, homology can be determined using, for example, the literature-based algorithm BLAST (see Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA (see Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0050] In one example, a polynucleotide comprising a specific nucleic acid sequence provided herein is understood to include not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the complementary polynucleotide can hybridize at a Tm value that can be appropriately adjusted by those skilled in the art depending on the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and can be analyzed under the conditions described below; such conditions are specifically described in known literature. Examples of such hybridization conditions include, but are not limited to, conditions under which genes with high complementarity of 60% or more, 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, 99.5% or more, or 99.9% or more hybridize with each other, while genes with lower complementarity do not hybridize with each other; and conditions for washing once, specifically two to three times, at salt concentrations and temperatures equivalent to those used in standard Southern hybridization, such as 60°C, 1xSSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1xSSC, and 0.1% SDS; or 68°C, 0.1xSSC, and 0.1% SDS. Hybridization requires that two nucleotides have complementary sequences, or mismatches between bases may be tolerated depending on the stringency of hybridization. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and is well known in the relevant art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0051] Introduction of the polynucleotide or vector can be performed by those skilled in the art using a known transformation method. As used herein, the term "transformation" refers to the process of introducing a specific polynucleotide or a vector containing the same into a host cell. The transformed polynucleotide can be integrated into a chromosome or extrachromosomally located within the host cell. For example, transformation can involve introducing a polynucleotide encoding a target protein (foreign protein) or a vector containing the same into a host cell to enable the expression of the protein encoded by the polynucleotide in the host cell. The polynucleotide can also include DNA and / or RNA encoding the target protein. The polynucleotide may be introduced in any form, as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes expression regulatory elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell. The term "operably linked" may mean that an expression regulatory element (e.g., a promoter) and a polynucleotide are functionally linked so that the expression regulatory element can regulate transcription (e.g., initiate transcription) of a polynucleotide encoding a target protein (foreign protein). Operable linkage can be achieved using recombinant DNA techniques known in the art, such as, but not limited to, conventional site-specific DNA cleavage and ligation.

[0052] The method for transforming the polynucleotide into a host cell can be any method for introducing nucleic acids into cells (microorganisms), and can be selected appropriately from transformation techniques known in the art depending on the host cell. Examples of known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran, cationic liposome, lipofection, and lithium acetate-DMSO.

[0053] The polynucleotide can be introduced (inserted) into the host cell genome (chromosome) using a known method appropriately selected by those skilled in the art, for example, using an RNA-guided endonuclease system (RNA-guided endonuclease system or CRISPR system; for example, one or more selected from the group consisting of (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding the same, or a vector containing the gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA)), a DNA encoding the same, or a vector containing the DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector containing both an RNA-guided endonuclease-encoding gene and a guide RNA-encoding DNA), etc.), but is not limited thereto.

[0054] As used herein, the term "vector" refers to a DNA product containing a polynucleotide sequence encoding a target protein operably linked to a suitable regulatory sequence so as to enable the expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and / or a sequence regulating the termination of transcription and / or translation. After being transformed into an appropriate host cell, the vector may be expressed independently of the host cell's genome (genetic body) or integrated into the host cell's genome.

[0055] The vectors usable herein are not particularly limited as long as they are replicable in host cells and may be selected from any commonly used vector. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, phage or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used. Plasmid vectors that can be used include pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specific examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0056] The vectors usable herein may be known expression vectors and / or vectors for inserting a polynucleotide into a host cell chromosome. The polynucleotide may be inserted into a host cell chromosome by any method known in the art, including, but not limited to, homologous recombination or the CRISPR system. The vector may additionally contain a selection marker to confirm the presence or absence of the insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the polynucleotide insertion. The selection marker can be selected from genes that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0057] Another example provides a composition for producing pantothenic acid or pantoic acid, comprising the microorganism.

[0058] Another example provides a method for producing pantothenic acid or pantoic acid, comprising culturing the microorganism in a medium.

[0059] Another example provides a method for increasing the ability of a microorganism to produce pantothenic acid and / or pantoic acid, or a method for imparting pantothenic acid production ability to the microorganism, the method comprising enhancing the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase in the microorganism.

[0060] The step of enhancing the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase in the microorganism can include introducing a mutation into the microorganism that causes it to express a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

[0061] The step of introducing the mutation may include a step of introducing (transforming) a polynucleotide encoding a polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase or a recombinant vector containing the polynucleotide into a microorganism.

[0062] Another example provides a method for producing pantothenic acid and / or pantoic acid, comprising culturing a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase in a medium, and the method may further comprise recovering pantothenic acid and / or pantoic acid from the cultured microorganism, the medium, or both after the culturing step.

[0063] In the above method, the step of culturing the microorganism can be carried out by known methods such as, but not limited to, batch culture, continuous culture, or fed-batch culture. The culture conditions can be adjusted to an appropriate pH (e.g., pH 5-9, specifically pH 6-8, most specifically pH 6.8) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), and aerobic conditions can be maintained by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature can be maintained at 20-45°C or 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited thereto. Pantothenic acid and / or pantoic acid produced by the culture can be secreted into the medium or remain intracellularly.

[0064] The media usable for the culture may include, but are not limited to, one or more carbon sources selected from the group consisting of sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), milk fats and fats (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), either individually or in combination. The nitrogen sources may include, but are not limited to, one or more nitrogen-containing organic compounds (e.g., peptone, yeast extract, broth, malt extract, corn steep liquor, soybean flour, and urea), and inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), either individually or in combination. The phosphorus source may be one or more selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts, either individually or in combination, but is not limited thereto. The medium may also contain essential growth-promoting substances such as other metal salts (e.g., magnesium sulfate or ferrous sulfate), amino acids, and / or vitamins.

[0065] The step of recovering pantothenic acid and / or pantoic acid may involve collecting the target amino acid from the culture medium, culture solution, or microorganism using a suitable method known in the art. For example, the recovery step may be carried out by one or more methods selected from centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method of recovering pantothenic acid and / or pantoic acid may additionally include a purification step before, simultaneously with, or after the recovery. [Effects of the Invention]

[0066] This specification provides a 3-methyl-2-ketobutanoate hydroxymethyltransferase mutant, and a technique for increasing the ability of a microorganism to produce pantothenic acid and / or pantoic acid using the mutant. By introducing a mutation that causes the microorganism to express the 3-methyl-2-ketobutanoate hydroxymethyltransferase mutant into the microorganism, a technique is provided that can improve the productivity of pantothenic acid and / or pantoic acid or impart the ability to produce pantothenic acid and / or pantoic acid. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be obvious to those skilled in the art that the examples described below can be modified within the scope of the essential gist of the invention.

[0068] Example 1. Searching and screening for 3-methyl-2-oxobutanoate hydroxymethyltransferase genes Using the gene (panB) encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum ATCC13032 as a query, we performed an NCBI BLAST search to identify candidate genes that are likely to have the activity of a gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase, as well as the microorganisms that possess these genes. Among these, we selected genes encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from microorganisms with a biosafety level of 1, and these are summarized in Table 1.

[0069] [Table 1]

[0070] Example 2. Preparation of Corynebacterium spp. microorganism into which 3-methyl-2-oxobutanoate hydroxymethyltransferase derived from a foreign microorganism has been introduced The genome of the microorganism isolated in Example 1 was extracted, and PCR was performed using the extracted genome as a template with the primer sequences listed in Table 1 to amplify a DNA fragment encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase. The PCR was performed using PfuUltra™ 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 DNA fragment (panB) encoding each 3-methyl-2-oxobutanoate hydroxymethyltransferase was obtained.

[0071] To obtain the PLM1 promoter derived from Corynebacterium glutamicum, PCR was performed in the same manner as described above using Corynebacterium glutamicum (ATCC13032) genomic DNA as a template and primers of SEQ ID NOs: 27 and 28 to obtain a promoter DNA fragment.

[0072] 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, was used with the resulting DNA fragments (panB and PLM1 promoters) at a molar concentration (M) of 2:1:1 (pECCG117 vector:panB:PLM1). Plasmids were obtained by cloning using the TaKaRa Infusion Cloning Kit according to the instructions provided. The names of the obtained plasmids and the information on the introduced genes are shown in Table 1.

[0073] The 13 constructed vectors were transformed into Corynebacterium glutamicum ATCC13032 by electroporation to generate strains expressing exogenous PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase).

[0074] Example 3. Investigation of the pantothenic acid productivity of Corynebacterium spp. microorganisms expressing 3-methyl-2-oxobutanoate hydroxymethyltransferase derived from foreign microorganisms To confirm the pantothenic acid productivity of the strains expressing panB derived from various exogenous microorganisms obtained in Example 2, the parent strain (non-transformed strain) and the strain were inoculated into 25 ml of a production medium having the following composition in a 250 ml corner-baffled flask, and cultured at 32°C for 48 hours with shaking at 200 rpm to produce pantothenic acid.

[0075] <Production medium> Glucose 10%, beta-alanine 0.5%, 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

[0076] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and the supernatant was diluted 1 / 10 with TDW (triple distilled water) and then analyzed by HPLC to measure the concentrations of pantothenic acid and L-valine. The results are shown in Table 2 below.

[0077] [Table 2]

[0078] As shown in Table 2, the parent strain, Corynebacterium glutamicum ATCC13032, did not produce pantothenic acid, whereas all tested Corynebacterium glutamicum strains expressing exogenous panB produced an average of approximately 0.6 g / L of pantothenic acid. In particular, among the exogenous PanB-expressing strains, the E. coli-derived panB-expressing strain ATCC13032 pECCG117-panB(EC) showed the highest pantothenic acid productivity (1.2 g / L).

[0079] The above results show that all of the enzymes (3-methyl-2-oxobutanoate hydroxymethyltransferases) derived from the 13 microorganisms selected in Example 1 exhibit pantothenic acid production ability, and among these, the enzyme derived from Escherichia coli has a particularly high pantothenic acid production ability.

[0080] Example 4. Preparation of Corynebacterium microorganisms into which the 3-methyl-2-oxobutanoate hydroxymethyltransferase gene derived from Escherichia coli has been introduced In Example 3, a plasmid was constructed to introduce the 3-methyl-2-oxobutanoate hydroxymethyltransferase-encoding gene (panB) derived from Escherichia coli, which was confirmed to have particularly excellent pantothenic acid-producing ability, into Corynebacterium glutamicum ATCC13032.

[0081] First, we constructed a vector to delete the panB gene present in the parent strain. PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and primers SEQ ID NOs: 29 and 30 and SEQ ID NOs: 31 and 32. 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 25 times. As a result, we isolated a 1000-bp gene fragment upstream of the panB gene and a 1000-bp gene fragment downstream of the panB gene. Each amplified product was purified using a QIAGEN PCR Purification kit and used as the insert DNA fragment for vector construction.

[0082] The pDZ vector (Korea Patent Registration No. 0924065) was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The vector and DNA fragments (a 1000 bp gene fragment upstream of the panB gene and a 1000 bp gene fragment downstream of the panB gene) were then mixed at a molar concentration (M) of 2:1:1 and cloned using the TaKaRa Infusion Cloning Kit according to the instructions provided. This produced the vector pDZ_ΔpanB for deleting the panB gene on the chromosome.

[0083] To prepare the panB gene derived from E. coli, PCR was performed using the plasmid pECCG117-panB(EC) prepared in Example 2 as a template and primers represented by SEQ ID NOs: 33 and 34. PCR consisted of 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, resulting in the isolation of a 1077-bp DNA fragment. The pDZ_ΔpanB vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the resulting DNA fragment were cloned at a molar concentration (M) of 1:2 using the Takara Infusion Cloning Kit according to the instructions provided with the kit to prepare the vector pDZ_ΔpanB::PLM1-panB(EC) for introducing the panB gene derived from E. coli into the chromosome.

[0084] The constructed vectors, pDZ_ΔpanB and pDZ_ΔpanB::panB(EC), were transformed into Corynebacterium glutamicum ATCC13032 by electroporation. After a second crossover, a strain lacking panB on the chromosome (ΔpanB strain) and a strain containing E. coli-derived panB (ΔpanB::panB(EC)) were obtained. The appropriate replacement of E. coli-derived panB was confirmed using the MASA (Mutant Allele Specific Amplification) PCR method (Takeda et al., Hum. Mutation, 2, 112–117 (1993)) with the following primer combinations: The primary determination was made by screening strains for amplification using primer combinations matching E. coli panB (SEQ ID NOs: 35 and 28, and SEQ ID NOs: 36 and 1), and the panB sequences of the selected strains were secondary confirmed by analysis using the primer combinations of SEQ ID NOs: 35 and 36.

[0085] To confirm the pantothenic acid productivity of the mutant strains obtained above, the wild-type strain, the ΔpanB strain, and the ΔpanB::panB(EC) mutant strain of Corynebacterium glutamicum ATCC13032 were inoculated into 250 ml corner-baffled flasks containing 25 ml of production medium (see Example 3) and cultured at 32°C for 48 hours with shaking at 200 rpm to produce pantothenic acid.

[0086] 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). HPLC analysis was then performed to measure the concentrations of pantothenic acid and L-valine. The results are shown in Table 3 below.

[0087] [Table 3]

[0088] As shown in Table 3, the wild-type and panB-deficient (ΔpanB) strains of Corynebacterium glutamicum ATCC13032 were unable to produce or barely produced any pantothenic acid, whereas the mutant Corynebacterium glutamicum expressing exogenous panB (ΔpanB::panB(EC)) produced pantothenic acid at a concentration of 0.4 g / L.

[0089] Example 5. Creation of random mutant strains by artificial mutagenesis (NTG-based mutation) and selection of panB-producing strains In this example, in order to obtain a microbial mutant with improved pantothenic acid production ability, the following method was used to induce a microbial mutation in the parent strain Corynebacterium glutamicum ATCC13032ΔpanB::panB(EC) mutant strain expressing Escherichia coli-derived panB, which was prepared in Example 4.

[0090] Specifically, Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC) strain was activated by culturing in activation medium for 16 hours, inoculated into seed medium sterilized at 121°C for 15 minutes, and cultured for 14 hours. Five ml of culture medium was harvested. The harvested culture medium was washed with 100 mM citrate buffer, treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) at a final concentration of 200 mg / L for 20 minutes, and then washed with 100 mM phosphate buffer. The NTG-treated strain was plated on minimal medium and the mortality rate was measured; the mortality rate was 85%. The surviving cells were inoculated into a production medium and cultured. Finally, two mutant strains exhibiting excellent pantothenic acid production were selected and named Corynebacterium glutamicum CJVB5-01 (Corynebacterium glutamicum, CJVB5-01) and CJVB5-02 (Corynebacterium glutamicum, CJVB5-02).

[0091] The composition of the medium used in this example is as follows:

[0092] <Activation medium> Beef extract 1%, polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2

[0093] <Production medium> Glucose 10%, beta-alanine 0.5%, 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

[0094] <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 200 μg / l, agar 2%, pH 7.2

[0095] To confirm the pantothenic acid-producing ability of the obtained mutant strains, Corynebacterium glutamicum CJVB5-01 and CJVB5-02, Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC) strain, the CJVB5-01 mutant strain, and the CJVB5-02 mutant strain were inoculated into 250 ml corner-baffled flasks containing 25 ml of production medium, and then cultured at 32°C for 48 hours with shaking at 200 rpm to produce pantothenic acid.

[0096] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and the supernatant was diluted 1 / 10 with TDW (triple distilled water) and then analyzed by HPLC to measure the concentrations of pantothenic acid and L-valine. The results are shown in Tables 4 and 5 below.

[0097] [Table 4]

[0098] [Table 5]

[0099] As shown in Tables 4 and 5, the Corynebacterium glutamicum ΔpanB strain did not produce pantothenic acid, and the Corynebacterium glutamicum mutants CJVB5-01 and CJVB5-02 exhibited superior pantothenic acid production compared to Corynebacterium glutamicum ΔpanB::panB(EC) in which an exogenous panB gene was inserted. Furthermore, the concentration of valine, a substance that utilizes 3-methyl-2-oxobutanoate as a substrate, decreased, confirming that the pantothenic acid production abilities of the CJVB5-1 and CJVB5-02 mutants were significantly greater than that of the wild type.

[0100] Genome sequencing of the Corynebacterium glutamicum CJVB5-01 mutant confirmed that the inserted E. coli panB gene was mutated to encode a mutant with a G116A mutation (a substitution of A (Ala) for G (Gly) at amino acid residue 116 in the amino acid sequence of SEQ ID NO: 37) introduced into wild-type E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase (SEQ ID NO: 37). Hereinafter, the designation of an amino acid mutation using an amino acid position, such as "G116A," is understood to refer to an amino acid mutation and / or a genetic mutation that induces such an amino acid mutation. The amino acid sequence of the E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant with the G116A mutation introduced is set forth in SEQ ID NO: 62.

[0101] Genome sequencing of the Corynebacterium glutamicum CJVB5-02 mutant confirmed that the inserted E. coli panB gene was mutated to encode a mutant with an A159L mutation (A (Ala, alanine) at the amino acid residue corresponding to the 159th residue in the amino acid sequence of SEQ ID NO: 37 was replaced with L (Leu, leucine)) introduced into the wild-type E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase (SEQ ID NO: 37). Hereinafter, the designation of an amino acid mutation using an amino acid position, such as "A159L," is understood to refer to an amino acid mutation and / or a genetic mutation that induces such an amino acid mutation. The amino acid sequence of the E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase mutant with the A159L mutation introduced is set forth in SEQ ID NO: 110.

[0102] The above results confirmed that the CJVB5-01 and CJVB5-02 mutant strains obtained by random mutation do not inhibit the pathway for synthesizing pantothenic acid from pyruvate and can produce pantothenic acid with high efficiency and high yield.

[0103] Example 6. Construction of a mutant panB plasmid with 3-methyl-2-ketobutanoate hydroxymethyltransferase activity In order to confirm that the amino acid residues corresponding to the 116th and / or 159th residues, which are the mutation positions of E. coli PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase) that were confirmed to affect pantothenic acid production in Example 5, are important positions for increasing pantothenic acid production, mutants were prepared in which the amino acid residues at these positions were substituted with other amino acids, and their effects were confirmed.

[0104] Using pECCG117-panB(EC) (see Table 1) prepared in Example 2 as a template and the primers listed in Table 6 below, mutants were prepared by introducing a random mutation (saturated mutagenesis) in which the amino acid G (Gly) at position 116 of E. coli PanB (SEQ ID NO: 37) was replaced with another amino acid (i.e., a panB gene mutated to encode E. coli PanB with the random mutation was introduced). Additionally, using the primers listed in Table 7 below, mutants were prepared by introducing a random mutation in which the amino acid A (Ala) at position 159 of E. coli PanB (SEQ ID NO: 37) was replaced with another amino acid. pECCG117-panB(EC) prepared in Example 1 was used as a template. The amino acids replaced by the mutant strains subjected to saturation mutagenesis and the primers used for each mutant are summarized in Tables 6 and 7 below.

[0105] [Table 6]

[0106] [Table 7]

[0107] Specifically, PCR was carried out using the primers shown in Tables 6 and 7 and pECCG117-panB(EC) (see Table 1) prepared in Example 2 as a template.TM PCR was performed using Pfu-X DNA polymerase (SolGent Co., Ltd.), with denaturation at 95°C for 10 minutes, followed by 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 5 minutes. As a result, a 610 bp DNA fragment of the 5' upstream region and a 470 bp DNA fragment of the 3' downstream region were obtained centered on the mutation (116th residue) of the 3-methyl-2-oxobutanoate hydroxymethyltransferase gene, and a 477 bp DNA fragment of the 5' upstream region and a 318 bp DNA fragment of the 3' downstream region were obtained centered on the 159th residue. 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 obtained DNA fragments (116th residue: 610 bp DNA fragment of the 5' upstream region and 470 bp DNA fragment of the 3' downstream region, 159th residue: 477 bp DNA fragment of the 5' upstream region and 318 bp DNA fragment of the 3' downstream region) were used in a molar concentration (M) of 2:1:1 using the Infusion Cloning Kit (TaKaRa). A plasmid for introducing the mutated panB gene was obtained by cloning according to the manual provided with the kit. To obtain the PLM1 promoter from Corynebacterium glutamicum, PCR was performed in the same manner as described above using Corynebacterium glutamicum (ATCC13032) genomic DNA as a template and primers with SEQ ID NOs: 108 and 109 to obtain a promoter DNA fragment.

[0108] The information on the mutant plasmids obtained is summarized in Tables 8 and 9 below.

[0109] [Table 8]

[0110] [Table 9]

[0111] Example 7. Evaluation of pantothenic acid production ability of mutant 3-methyl-2-ketobutanoate hydroxymethyltransferase The mutant plasmids (Tables 8 and 9) and pECCG117-panB(WT-EC) (Table 1) prepared in Example 6 were introduced into the ATCC13032ΔpanB strain prepared in Example 4 by electroporation, and then the strains were plated on a selective medium containing 25 mg / L kanamycin to obtain a total of 19 transformed mutant strains into which random mutations (saturated mutagenesis) had been introduced. Then, flask evaluation was carried out in the same manner as in Example 3, and the resulting pantothenic acid productivity was measured. The results are shown in Tables 10 and 11.

[0112] [Table 10]

[0113] As can be seen from Table 10 above, the ATCC13032ΔpanB strain did not produce pantothenic acid, whereas all of the mutant strains incorporating E. coli PanB (wild-type) or its mutants exhibited pantothenic acid production. Furthermore, the mutant strains incorporating the G116S, G116C, G116L, G116I, G116T, G116V, G116D, G116E, G116N, G116A, G116M, or G116Q mutations produced higher levels of pantothenic acid than the ATCC13032ΔpanB pECCG117-panB(WT) mutant strain containing E. coli PanB (wild-type). These results confirmed that both wild-type and mutant forms of E. coli PanB have the effect of increasing pantothenic acid production, and that the 116th amino acid residue of PanB (SEQ ID NO: 37) is particularly important for pantothenic acid production, and that substituting various amino acids for this amino acid position further increases pantothenic acid production.

[0114] In this example, the ATCC13032ΔpanB pECCG117-panB(G116A) strain (designated Corynebacterium glutamicum CV03-5001), which was confirmed to have the best pantothenic acid production ability, was deposited with the Korea Microorganism Collection (KMC), Hongje-dong, Seodaemun-gu, Seoul, Republic of Korea, on June 8, 2020, and was assigned the deposit number KCCM12744P.

[0115] [Table 11]

[0116] Furthermore, as can be seen from Table 11, the wild-type Corynebacterium glutamicum ATCC13032 strain does not produce pantothenic acid, whereas all of the 19 mutant strains prepared exhibited pantothenic acid production ability. Among these, the mutant strains in which the amino acid corresponding to residue 159 of wild-type panB (SEQ ID NO: 37) was mutated to arginine (R), serine (S), tyrosine (Y), cysteine ​​(C), proline (P), histidine (H), leucine (L), isoleucine (I), threonine (T), lysine (K), valine (V), methionine (M), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q) produced pantothenic acid at a higher level than the ATCC13032 pECCG117-panB(WT) strain containing wild-type panB. These results confirmed that the amino acid residue corresponding to residue 159 of panB (SEQ ID NO: 37) is an important position for pantothenic acid production, and that substituting various amino acids different from the original amino acid at this position further increases the ability to produce pantothenic acid.

[0117] In this example, the ATCC13032ΔpanB pECCG117-panB(A159L) strain, which was confirmed to have the best pantothenic acid production ability, was named CV03-5002 and deposited at the Korea Microorganism Collection, Hongje-dong, Seodaemun-gu, Seoul, Republic of Korea, on April 13, 2021, and was assigned the deposit number KCCM12973P.

[0118] Example 8. Construction and evaluation of mutant 3-methyl-2-ketobutanoate hydroxymethyltransferase with improved pantothenic acid production ability The 3-methyl-2-ketobutanoate hydroxymethyltransferase G116A mutant, the effect of which was confirmed in Example 7, was combined with the 3-methyl-2-ketobutanoate hydroxymethyltransferase A159L mutant to confirm whether there was any additional improvement in activity.

[0119] To construct a vector containing the G116A and A159L mutations, the same vector containing both G116A and A159L as described in Example 2 was constructed using the pECCG117-panB(A159L) constructed in Example 2 as a template and primers set forth in SEQ ID NOs: 74, 126, and 77, 127. The constructed mutant plasmid pECCG117-panB(G116A, A159L) was introduced into wild-type Corynebacterium glutamicum ATCC 13032 by electroporation and then plated on selective medium containing 25 mg / L kanamycin to obtain each transformant. The resulting pantothenic acid production was measured using a flask assay similar to that in Example 2. The results are shown in Table 12 below.

[0120] [Table 12]

[0121] As can be seen from Table 12, the ATCC13032 strain does not produce pantothenic acid, whereas the mutant strain containing both the amino acid mutations corresponding to residues 116 and 159 of panB produced pantothenic acid at a higher level than the ATCC13032 pECCG117-panB(WT) strain containing wild-type panB, and at a higher level than the mutant strains containing the A159L or G116A mutations. These results confirm that the amino acid residues corresponding to residues 116 and 159 of panB are important positions for pantothenic acid production, and that substituting amino acids at these positions with amino acids other than the original amino acid further increases pantothenic acid production.

[0122] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented 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 not limiting. The scope of the present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts.

[0123] (Accession number) Depository institution: Korea Microorganism Collection Center Accession number: KCCM12744P Date of acceptance: 20200608 Depository institution: Korea Microorganism Collection Center Accession number: KCCM12973P Date of acceptance: 20210413 JPEG0007730378000015.jpg240168JPEG0007730378000016.jpg240168

Claims

1. A polypeptide comprising an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 37, the amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with serine (S), cysteine ​​(C), proline (P), leucine (L), isoleucine (I), threonine (T), valine (V), methionine (M), aspartic acid (D), asparagine (N), or glutamine (Q); A polypeptide having the activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase.

2. The polypeptide of claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 110, 112, 114, 115, 117, 118, 120, 121, 122, 124 and 125.

3. In the amino acid sequence of SEQ ID NO: 37:

2. The polypeptide of claim 1, wherein the amino acid corresponding to the 116th residue from the N-terminus is additionally substituted with alanine (A), asparagine (N), threonine (T), glutamic acid (E), serine (S), valine (V), isoleucine (I), leucine (L), aspartic acid (D), cysteine ​​(C), glutamine (Q), or methionine (M).

4. The polypeptide of claim 3, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

128.

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

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A method for producing a recombinant vector comprising at least one selected from the group consisting of the polypeptide according to any one of claims 1 to 5, a polynucleotide encoding said polypeptide, and a recombinant vector comprising said polynucleotide. A microorganism that produces pantothenic acid or pantoic acid, A microorganism that is a Corynebacterium or Escherichia microorganism.

8. The microorganism according to claim 7, wherein the microorganism is Corynebacterium glutamicum.

9. A composition for producing pantothenic acid or pantoic acid, comprising the microorganism according to claim 7 or 8.

10. A method for producing pantothenic acid or pantoic acid, comprising culturing the microorganism according to claim 7 or 8 in a medium.

11. 11. The method for producing pantothenic acid or pantoic acid according to claim 10, further comprising, after the culturing step, recovering pantothenic acid or pantoic acid from the cultured microorganism, the medium, or both.

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