KR20240105656A - Microorganism having enhanced activity of anion symporter and uses thereof - Google Patents

Abstract

The present application provides a microorganism with enhanced anion transport protein 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, The production capacity of pantothenic acid and/or pantoic acid is excellent.

Description

Microorganism with enhanced activity of anion transport protein and uses thereof {Microorganism having enhanced activity of anion symporter and uses thereof}

Microorganisms with enhanced anion transport protein activity, a composition for producing pantothenic acid and/or pantoic acid containing the microorganisms, and a method for producing pantothenic acid and/or pantoic acid comprising culturing the microorganisms are provided.

Pantothenic acid is a substance belonging to the vitamin B complex and is also called vitamin B5. It is one of the commercially important substances with various applications in cosmetics, medicine, human nutrition, and animal nutrition. Pantothenic acid is a structure in which beta-alanine is linked to pantonic acid through an amide bond.

Pantothenic acid or pantoic acid can be manufactured chemically synthetically or biotechnologically by fermenting suitable microorganisms in a suitable medium. The advantage of biotechnological production methods using microorganisms is that the desired stereo-isomeric D-form of pantothenic acid or pantoic acid is formed.

Accordingly, there is a need for the development of microorganisms that have an advantageous effect in biotechnologically producing pantothenic acid and/or pantoic acid and technology for producing pantothenic acid and/or pantoic acid with high efficiency using the same.

(Prior Document 1) U.S. Patent No. 7718205

An example of the present application provides a microorganism that produces pantothenic acid or pantoic acid with enhanced anion transport protein activity.

Another example of the present application provides a method for producing pantothenic acid or pantoic acid comprising culturing the microorganism in a medium.

Another example of the present application provides pantothenic acid or a composition for producing pantosan containing the above microorganisms.

Another example of the present application provides the use of said microorganism for the production of pantothenic acid or pantoic acid.

Another example of the present application provides the use of the microorganism for the production of pantothenic acid or a composition for producing pantoic acid.

One aspect provides pantothenic acid or a microorganism producing pantoic acid with enhanced activity of anion transport proteins.

As used herein, the term “anion transport protein” may include without limitation any protein that has the function of transporting anions into cells. The anion transport protein may refer to a polypeptide or protein having an anion transport protein activity.

The anion may be a monovalent anion, a divalent anion, a trivalent anion, or a tetravalent anion.

In one example, the anion may be the conjugate base of an organic acid. The organic acid may be carboxylic acid, sulfinic acid, sulfonic acid, phenol, thiophenol, oxime, enol, imide, aromatic sulfonamide, primary nitro compound or secondary nitro compound, but is not limited thereto. In one example, the anion may be the conjugate base of carboxylic acid. The carboxylic acid may be, but is not limited to, methanoic acid, ethenoic acid, lactic acid, amino acid, citric acid, fatty acid (e.g., acetic acid, propionic acid, butanoic acid, palmitic acid, or oleic acid), or aromatic carboxylic acid (e.g., benzoic acid, salicylic acid). That is not the case. The carboxylic acid may include dicarboxylic acid, tricarboxylic acid, etc. containing two or more carboxylic acids. The dicarboxylic acid may be oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, serberic acid, azelaic acid, sebacic acid, phthalic acid, isophthalic acid, or terephthalic acid, but is not limited thereto. The carboxylic acid may be C1 carboxylic acid, C2 carboxylic acid, C3 carboxylic acid, C4 carboxylic acid, C5 carboxylic acid, or C6 carboxylic acid, but is not limited thereto.

The anion transport protein may have the activity of transporting the anion and the cation such as sodium ion or potassium ion into the cell.

The anion transport protein may have the activity of transporting divalent anions and sodium ions into cells.

The anion transport protein may be a DASS (Divalent anion:Na+ symporter) family-coupled anion transport protein (DASS family sodium-coupled anion symporter).

In one example, the anion transport protein may be the Ncam0154 protein.

In one example, the activity of the anion transport protein may be enhanced by introducing an anion transport protein or a polynucleotide encoding it, but is not limited thereto, and various methods well known in the art can be applied. In one example, the anion transport protein may be a protein native to the microorganism to be introduced or a foreign protein.

In the present specification, introduction of a foreign protein and/or gene refers to a microorganism belonging to a different genus (host cell), a microorganism belonging to a different species (host cell), or the same species as the microorganism into which the protein and/or gene is introduced (host cell). It may mean the introduction of proteins and/or genes derived from other microorganisms (host cells).

The anion transport protein and/or the gene encoding it may be derived from Corynebacterium stasis, but is not limited thereto. The sequence of the anion transport protein derived from Corynebacterium stationanis can be obtained from NCBI, a known database (for example, WP_066797267.1 or WP_075723752.1). In one example, the anion transport protein derived from Corynebacterium stasis may be the Ncam0154 protein.

In one example, the anion transport protein from Corynebacterium stasis has, includes, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 23. (essentially consisting of) can.

In one example, the anion transport protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 23. % or more, 97.19% or more, 97.37% or more, 97.56% or more, 97.75% or more, 97.94% or more, 98% or more, 98.12% or more, 98.31% or more, 98.50% or more, 98.69% or more, 98.87% or more, 99% or more , contains or consists of an amino acid sequence having 99.06% or more, 99.25% or more, 99.44% or more, 99.62% or more, 99.5% or more, 99.7% or more, 99.81% or more, or 99.9% or more sequence homology or sequence identity. You can. In addition, if a protein has such homology or identity and exhibits anion transport protein activity, a variant of the anion transport protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the anion transport protein. there is. For example, addition or deletion of sequences at the N-terminus, C-terminus and/or within the amino acid sequence that do not alter anion transport protein activity, mutations that may occur naturally, silent mutations or conservative substitutions. This is the case with .

The “conservative substitution” means replacing one amino acid with another amino acid having similar structural and/or chemical properties. These amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

The anion transport protein gene may refer to a polynucleotide encoding the anion transport protein. The polynucleotide encoding the anion transport protein can be prepared based on the amino acid sequence of the anion transport protein, codon information known in the art, and codons preferred in the host microorganism (cell) to be introduced, and in one example, corynebacterium It may be from Bacterium stasis, but is not limited thereto.

In one example, the polynucleotide encoding (encoding) an anion transport protein derived from Corynebacterium stasis has, includes, consists of, or consists of the nucleic acid sequence of SEQ ID NO: 13, or the nucleic acid It may essentially consist of a sequence.

In one example, the anion transport protein derived from Corynebacterium stasis has, includes, consists of, or consists of the nucleic acid sequence of SEQ ID NO: 13, or is produced by a polynucleotide consisting essentially of the nucleic acid sequence. It may be coded.

In one example, the polynucleotide encoding the anion transport protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the nucleic acid sequence of SEQ ID NO: 13. %, 99.5%, 99.7%, or 99.9% or more sequence homology or sequence identity may include or consist of a nucleic acid sequence.

In the present application, the term "polynucleotide or polypeptide" refers to "having, comprising, consisting of, or consisting essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence". This may mean that a nucleotide or polypeptide essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and the specific nucleic acid sequence (base sequence) or amino acid sequence may be used to It can be interpreted as including (or not excluding) a “substantially equivalent sequence” in which mutations (deletions, substitutions, modifications, and/or additions) are made to the nucleic acid sequence (base sequence) or amino acid sequence. . In one example, a polynucleotide or polypeptide "has, includes, consists of, or consists essentially of a specific nucleic acid sequence (base sequence) or amino acid sequence" means that the polynucleotide or polypeptide is (i) essentially comprising the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) at least 70%, 80%, 85%, or 90% of the specific nucleic acid sequence (base sequence) or amino acid sequence. or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more. It may mean that it consists of or essentially includes a nucleic acid sequence or amino acid sequence having homology or identity and maintains its original function and/or desired function. In one example, the desired function may mean a function that increases (improves) or imparts the pantothenic acid and/or pantoic acid production ability of microorganisms.

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

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may be used with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of a sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization of polynucleotides with polynucleotides containing common codons or codons taking codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: It can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Or, as performed in the Needleman program in 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), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (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), or BLAST from the National Center for Biotechnology Information Database. ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity or identity of polynucleotides or polypeptides is defined in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. This can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the number of similarly aligned symbols (i.e. nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are (1) a binary comparison matrix (containing values 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

The polynucleotide encoding the anion transport protein can be introduced into a microorganism using a recombinant vector, but is not limited thereto. The recombinant vector can be used as an insertion vector or an expression vector. Expressing the anion transport protein in a microorganism can be performed by culturing a recombinant microorganism containing a polynucleotide encoding the anion transport protein or a recombinant vector containing the same.

The introduction of a polynucleotide encoding the anion transport protein or a recombinant vector containing the same into a microorganism can be performed by a person skilled in the art by appropriately selecting a known transformation method. The transformation method can be performed by any method of introducing a nucleic acid into a host cell (microorganism), and can be performed by appropriately selecting transformation techniques known in the art depending on the host cell. The known transformation methods include electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), Examples may include the DEAE-dextran method, cationic liposome method, lipofection, and lithium acetate-DMSO method, but are not limited thereto.

As long as the transformed polynucleotide can be expressed within the host cell, it may be inserted into and located within the chromosome of the host cell or may be located outside the chromosome. As long as the polynucleotide can be introduced and expressed into a host cell, the form in which it is introduced is not limited. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include expression control elements such as a promoter, transcription termination signal, ribosome binding site, and/or translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell. As used herein, the term "operably linked" may mean that an expression control element (e.g., promoter) and a polynucleotide are functionally connected to perform transcriptional regulation (e.g., transcription initiation) of the polynucleotide. . Operable linkage can be accomplished using genetic recombination techniques known in the art.

As used in this application, the term “vector” refers to a DNA preparation for delivering a polynucleotide of interest into a suitable host or host cell. As an example, it may include a base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself. For example, a polynucleotide of interest can be inserted into a chromosome through a vector for chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. The selection marker is used to select cells transformed with a vector, that is, to confirm the insertion of the target nucleic acid molecule, and is used to determine selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers that give may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

The vector used in this 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 vectors or cosmid vectors, and pDZ-, pDC-, and pBR-based plasmid vectors can be used. , pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. For example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

In this application, the term “enhancement” of polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase may include showing an activity that it did not originally have, or showing improved activity compared to the intrinsic activity or activity before modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the change in trait when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “activity before modification.” “Enhanced,” “upregulated,” “overexpressed,” or “increased” in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation. It means improved compared to (expression amount).

The enhancement can be achieved by introducing an exogenous polypeptide, enhancing the activity and/or increasing the concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed from the level of activity of the polypeptide, the expression level, or an increase in the amount of products due to the activity of the polypeptide.

Enhancement of the activity of the polypeptide can be done by applying 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 microorganism before modification. Specifically, genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology, may be used, but are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology 2010, Vol. 2. 1-16, Molecular Cloning 2012, etc.

Specifically, the reinforcement of the polypeptide of the present application is

1) Increased intracellular copy number of the polynucleotide encoding the polypeptide;

2) Replacement of the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

3) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

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

6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) Codon optimization of the polynucleotide encoding the polypeptide;

8) Analyzing the tertiary structure of the polypeptide, select exposed sites and modify or chemically modify them; or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited.

More specifically,

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

2) Replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a highly active sequence, for example, deletion, insertion, or non-conservative use to further enhance the activity of the expression control region. Alternatively, it may be a mutation in the sequence due to conservative substitution or a combination thereof, or replacement with a sequence with stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but the method is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US Patent US 7662943 B2), 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 US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) The base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon with a higher polypeptide expression rate than the internal start codon. It may be a substitution by sequence, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) includes deletion, insertion, non-conservative or conservative amino acid sequence of the polypeptide or polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide. It may be a mutation in the sequence due to a substitution or a combination thereof, or a replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to increase activity, but is limited thereto. no. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to check whether chromosome insertion has occurred.

6) Introduction of a foreign polynucleotide showing the activity of the polypeptide may be introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same/similar activity as the polypeptide. There are no restrictions on the origin or sequence of the foreign polynucleotide as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and by expressing the introduced polynucleotide in the host cell, a polypeptide can be produced and its activity can be increased.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the native polynucleotide to increase transcription or translation within the host cell, or codon optimization of the foreign polynucleotide to increase transcription or translation within the host cell. The codon may have been optimized to achieve this.

Above 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to modify or chemically modify the sequence information, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins to determine sequence similarity. Depending on the degree, a template protein candidate may be determined, the structure may be confirmed based on this, and an exposed site to be modified or chemically modified may be selected and modified or modified.

Such enhancement of polypeptide activity means that the activity or concentration of the corresponding polypeptide is increased based on the activity or concentration of the polypeptide expressed in the wild type or unmodified microbial strain, or the amount of product produced from the polypeptide is increased. may be increased, but is not limited to this.

In this application, the term “weakening” of a polypeptide is a concept that includes both reduced activity or no activity compared to the intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

The weakening occurs when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to mutation of the polynucleotide encoding the polypeptide, inhibition of expression of the gene of the polynucleotide encoding the polypeptide, or polypeptide When the overall polypeptide activity level and/or concentration (expression level) in the cell is lower than that of the native strain due to inhibition of translation into peptide, etc., when the polynucleotide is not expressed at all, and/or Even if the polynucleotide is expressed, the case where the polypeptide is not active may also be included. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild type, or unmodified microorganism before the change in trait when the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “activity before modification.” The fact that the activity of a polypeptide is “inactivated, deficient, reduced, down-regulated, lowered, or attenuated” compared to the intrinsic activity means that the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation was lowered. do.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by applying various methods well known in the art (e.g., Nakashima N et al. , Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Molecular Cloning 2012, etc.

Specifically, the weakening of the polypeptide

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

2) Modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion/substitution/addition of one or more amino acids on the amino acid sequence) such that the activity of the polypeptide is eliminated or weakened;

4) Modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is removed or weakened (e.g., the nucleic acid base sequence of the polypeptide gene to encode a polypeptide modified to eliminate or weaken the activity of the polypeptide) deletion/substitution/addition of one or more nucleotide bases);

5) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

6) Introduction of an antisense oligonucleotide (eg, antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide;

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which ribosome attachment is impossible;

8) Addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited.

for example,

1) Deletion of part or all of the gene encoding the polypeptide may be removal of the entire polynucleotide encoding the target polypeptide endogenous in the chromosome, replacement with a polynucleotide in which some nucleotides have been deleted, or replacement with a marker gene. .

In addition, the above 2) modification of the expression control region (or expression control sequence) is a mutation in the expression control region (or expression control sequence) caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or a weaker mutation. It may be replacement with an active sequence. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

In addition, 3) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide, for example, encodes another start codon with a lower polypeptide expression rate compared to the internal start codon. It may be a substitution with a base sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) may include deletion, insertion, non-conservative or non-conservative modification of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide. It may be a mutation in the sequence due to conservative substitution or a combination thereof, or a replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity, or an amino acid sequence or polynucleotide sequence that has been improved to have no activity, but is limited thereto. no. For example, gene expression can be inhibited or weakened by introducing mutations in the polynucleotide sequence to form a stop codon, but is not limited to this. The "stop codon" is a codon that acts as a signal to signal the end of the protein synthesis process without specifying an amino acid among codons on mRNA. Generally, three types of stop codons, UAA, UAG, and UGA, can be used. .

6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecule. tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) In order to form a secondary structure to which ribosomes cannot attach, the addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide is mRNA. This may make translation impossible or slow it down.

8) The addition of a promoter transcribed in the opposite direction (Reverse transcription engineering, RTE) to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide is complementary to the transcript of the gene encoding the polypeptide. It may be that the activity is weakened by creating antisense nucleotides.

The microorganism of the present application may be a microorganism (e.g., a recombinant microorganism) that has been genetically modified through a vector to enhance the activity of the anion transport protein or the gene encoding it, but is not limited thereto. The vector is the same as described above.

In this application, the term “microorganism (or strain)” may include both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. The microorganism is a microorganism in which a specific mechanism is strengthened or weakened due to reasons such as insertion of an external gene or strengthening or weakening of the activity of an intrinsic gene, and the desired polypeptide, protein, or product (e.g., pantothenic acid and/or pantoic acid) It may be a microorganism containing genetic modification for the production of.

The microorganism (or strain, recombinant cell) of the present application may be a microorganism that has the ability to produce (or produce) pantothenic acid and/or pantoic acid, or may have an improved (or increased) ability to produce pantothenic acid and/or pantoic acid.

The microorganism of the present application is a microorganism that does not naturally have the ability to produce pantothenic acid and/or pantoic acid, or an anion transport protein or a gene encoding the same is introduced into a microorganism that has the ability to produce pantothenic acid and/or pantoic acid, or is enhanced to produce pantothenic acid and/or Alternatively, it may be a microorganism endowed with or improved in the ability to produce pantoic acid, but is not limited thereto. The microorganism endowed or improved with pantothenic acid and/or pantoic acid production ability may be a microorganism into which an anion transport protein or a gene encoding the same has been introduced.

That the microorganism has an increased (improved) ability to produce pantothenic acid and/or pantoic acid or has the ability to produce pantothenic acid and/or pantoic acid means that the microorganism is an unmodified microorganism, a cell before recombination, a parent strain, a wild-type strain, and/or another microorganism. Unmodified microorganisms with improved pantothenic acid and/or pantoic acid production ability or no pantothenic acid and/or pantoic acid production ability, cells before recombination, parent strain and/or wild type strain than microorganisms into which the derived anion transport protein or the gene encoding the same has been introduced. Unlike, it may mean that the ability to produce pantothenic acid and/or pantoic acid is granted.

One aspect provides pantothenic acid or a microorganism producing pantoic acid with enhanced activity of an anion transport protein.

According to one example, a microorganism in which an anion transport protein or a gene encoding it is introduced or its activity is enhanced has an improved ability to produce pantothenic acid and/or pantoic acid compared to a microorganism before introduction or a microorganism before enhancement, that is, an unmodified microorganism of the same type. You can. In this application, “unmodified microorganism” does not exclude strains containing mutations that may occur naturally in microorganisms, and is a wild-type strain or a natural strain itself, or whose characteristics change due to genetic mutation caused by natural or artificial factors. It may refer to a strain before becoming a strain. For example, the unmodified microorganism may refer to a strain in which an anion transport protein or a gene encoding the same is not introduced, its activity is not enhanced, or its activity is not enhanced. The “non-modified microorganism” may be used interchangeably with “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated strain”, “non-modified strain”, “non-mutated microorganism” or “reference microorganism”. As described above, the activity of the anion transport protein or the gene encoding it is enhanced.

In one example, in a microorganism into which an anion transport protein or a gene encoding the same is introduced or its activity is enhanced, the anion transport protein may be derived from Corynebacterium stasis, but is not limited thereto as long as it exhibits the same activity. The anion transport protein is the same as previously described.

In one example, the microorganism may be a microorganism with additionally enhanced activity of 3-methyl-2-oxobutanoate hydroxy methyltransferase.

In one example, the 3-methyl-2-oxobutanoate hydroxy methyltransferase may be derived from E. coli.

In one example, the microorganism may be a microorganism into which an exogenous 3-methyl-2-oxobutanoate hydroxy methyltransferase or a gene encoding the same has been introduced.

In one example, the microorganism may be a microorganism into which 3-methyl-2-oxobutanoate hydroxy methyltransferase derived from E. coli or a gene encoding the same has been introduced.

The 3-methyl-2-oxobutanoate hydroxy methyltransferase may be the panB protein.

In one example, the target strain to compare whether the pantothenic acid and/or pantoic acid production ability is increased is the wild-type Corynebacterium glutamicum ATCC13032 strain or the 3-methyl-2-oxobutanoate hydroxy methyltransferase. It may be a microorganism with enhanced activity (e.g., a microorganism of the genus Corynebacterium, Corynebacterium glutamicum, or Corynebacterium glutamicum ATCC13032 strain), but is not limited thereto.

The microorganism may additionally contain a mutation that increases pantothenic acid and/or pantoic acid production, and the location of the mutation and/or the type of gene and/or protein subject to the mutation increase pantothenic acid and/or pantoic acid production. If possible, it can be included without limitation. The recombinant cells can be used without limitation as long as they are cells capable of transformation.

The microorganism may be a Corynebacterium sp. microorganism. The microorganisms in the Corynebacterium genus include Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, and Corynebacterium deser. Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans ), Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and It may be one or more types of microorganisms selected from the group consisting of Corynebacterium flavescens, but is not limited thereto.

As an example, the microorganism with improved pantothenic acid and/or pantoic acid production ability is compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which an anion transport protein from another microorganism or a gene encoding the same has been introduced. Local production capacity is newly granted, or about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more. or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more It may be an increase of about 900% or more, about 1,000% or more, about 1,500% or more, about 2,000% or more, about 2,500% or more, or about 3,000% or more, but is not limited thereto.

In another example, the microorganism with improved pantothenic acid and/or pantoic acid production ability is compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which an anion transport protein derived from another microorganism or a gene encoding the same has been introduced. Local production capacity is about 1.1 times more, about 1.2 times more, about 1.3 times more, about 1.4 times more, about 1.5 times more, about 1.6 times more, about 1.7 times more, about 1.8 times more, about 1.9 times more, about 2 times more. More than twice, about 2.5 times more, about 3 times more, about 4 times more, about 5 times more, about 6 times more, about 7 times more, about 8 times more, about 9 times more, about 10 times more, about 15 times more It may be 2 times or more, about 20 times more, about 25 times more, or about 30 times more (the upper limit is not particularly limited, for example, may be about 1,000 times or less), but is not limited thereto.

In another example, the microorganism with improved pantothenic acid and/or pantoic acid production ability is compared to the parent strain before mutation, an unmodified microorganism, or a microorganism into which an anion transport protein derived from another microorganism or a gene encoding the same has been introduced. Local production capacity is about 0.1 g/L or more, about 0.2 g/L or more, about 0.3 g/L or more, about 0.4 g/L or more, about 0.5 g/L or more, about 0.6 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, about 1 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, About 1.5 g/L or more, about 1.6 g/L or more, about 1.7 g/L or more, about 1.8 g/L or more, about 1.9 g/L or more, about 2.0 g/L or more, about 2.5 g/L or more, about 3 g/L or more, about 3.5 g/L or more, about 4 g/L or more, about 4.1 g/L or more, about 4.2 g/L or more, about 4.3 g/L or more, about 4.4 g/L or more, about 4.5 g/ L or more, about 4.6 g/L or more, about 4.7 g/L or more, about 4.8 g/L or more, about 4.9 g/L or more, about 5 g/L or more, about 5.5 g/L or more, about 6 g/L or more, About 7g/L or more, about 8g/L or more, about 9g/L or more, about 10g/L or more, about 15g/L or more, about 20g/L or more, about 25g/L or more, about 30g/L or more (the upper limit is There is no particular limitation, for example, it may be about 100 g/L or less), but it is not limited thereto.

The term “about” is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value that appears after the term “about.” Not limited.

Another aspect provides a method for producing (or producing) pantothenic acid and/or pantoic acid, comprising culturing the microorganism of the present application in a medium.

The method for producing pantothenic acid and/or pantoic acid of the present application may include culturing the microorganism of the present application in a medium.

In the present application, “cultivation” means growing a microorganism into which the anion transport protein of the present application or the gene encoding the same is introduced or the activity of which is enhanced, such as a Corynebacterium glutamicum strain, under appropriately controlled environmental conditions. . The culture process of the present application can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

In the present application, "medium" is mainly composed of nutrients necessary for culturing microorganisms into which the anion transport protein of the present application or the gene encoding it has been introduced or whose activity has been enhanced, such as Corynebacterium glutamicum strain. It refers to mixed substances and supplies water, nutrients and growth factors that are essential for survival and development. Specifically, the medium and other culture conditions used for cultivating the microorganisms of the present application can be any medium used for cultivating ordinary microorganisms without particular restrictions, but the microorganisms of the present application can be grown with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins while controlling temperature, pH, etc.

Specifically, culture media for microorganisms of the present application, such as strains of the genus Corynebacterium, can be found in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.Corynebacterium, USA, 1981). You can.

In the present application, the carbon source includes 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. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses (e.g. blackstrap molasses), rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. reducing sugars). Carbohydrates such as molasses (converted into molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, Organic nitrogen sources such as malt extract, corn steep liquor, casein hydrolyzate, fish or decomposition products thereof, defatted soybean cake or decomposition products thereof, etc. may be used. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

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. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.

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

Pantothenic acid and/or pantoic acid produced by the culture of the present application may be secreted into the medium or remain within the cells.

The method for producing pantothenic acid and/or pantoic acid of the present application includes preparing the microorganism of the present application, preparing a medium for culturing the microorganism, or a combination thereof (regardless of order, in any order), e.g. For example, it may be additionally included before the culturing step.

The method for producing pantothenic acid and/or pantoic acid of the present application adds the step of recovering pantothenic acid and/or pantoic acid from the culture medium (medium in which the culture was performed) or microorganisms (e.g., Corynebacterium genus strains). It can be included as . The recovering step may be additionally included after the culturing step.

The recovery involves collecting the desired pantothenic acid and/or pantoic acid using a suitable method known in the art according to the cultivation method of the microorganism of the present application, such as a batch, continuous or fed-batch culture method. It could be. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatographies such as chromatography, HPLC, or a combination of these methods can be used, and the desired pantothenic acid and/or pantoic acid can be recovered from the medium or microorganism using a suitable method known in the art.

In addition, the method for producing pantothenic acid and/or pantoic acid of the present application may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the method for producing pantothenic acid and/or pantoic acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step are performed sequentially or discontinuously regardless of the order, simultaneously, or in one step. It may be performed in an integrated manner, but is not limited thereto.

Another aspect is to provide pantothenic acid and/or a composition for producing pantoic acid containing the microorganism of the present application, a medium culturing the microorganism, or a combination thereof.

Another aspect provides the use of the microorganism for the production of pantothenic acid or pantoic acid.

Another aspect provides the use of the microorganism for the production of pantothenic acid or a composition for producing pantoic acid.

The compositions of the present application may further comprise any suitable excipients commonly used in pantothenic acid and/or compositions for the production of pantoic acid, such as preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents. Or it may be an isotonic topic, but it is not limited thereto.

When cultivating microorganisms with increased activity of the anion transport protein of the present application, high yield production of pantothenic acid and/or pantoic acid is possible. Accordingly, from an industrial perspective, convenience of production and reduction of manufacturing costs can be expected.

Hereinafter, the present invention will be described in more detail through the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Creation of pantothenic acid producing strains and search and selection of genes encoding anion transport proteins

Wild-type Corynebacterium glutamicum has the ability to produce D-pantothenic acid, but hardly produces it. Therefore, in order to easily select genes that increase the ability to excrete D-pantothenic acid according to the purpose of the present application, a strain with increased D-pantothenic acid production ability was used.

Specifically, in order to confirm the effect of the anion transport protein on increasing the productivity of pantothenic acid, the mutant strain Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC) (European Patent Publication EP 4032977 A1) was used as the parent strain, which has the ability to transport pantothenic acid. Overexpression of various anion transport proteins, which are presumed to increase production capacity, was induced. Microorganisms possessing candidate anion transport proteins, the corresponding protein sequences, and the nucleic acid sequences encoding them are summarized in Table 1 below.

microbe primer
order plasmid Nucleic acid sequence protein sequence One Corynebacterium stationis SEQ ID NO: 1, 2 pECCG117-Ncam0154 SEQ ID NO: 13 SEQ ID NO: 14 2 Blastococcus saxobsidens SEQ ID NO: 3, 4 pECCG117-Transporter(Bsa) SEQ ID NO: 15 SEQ ID NO: 19 3 Kibdelosporangium aridum SEQ ID NO: 5, 6 pECCG117-Transporter(Kar) SEQ ID NO: 16 SEQ ID NO: 20 4 Rhodococcus opacus SEQ ID NO: 7, 8 pECCG117-Transporter(Rop) SEQ ID NO: 17 SEQ ID NO: 21 5 Stackebrandtia nassauensis SEQ ID NO: 9, 10 pECCG117-Transporter(Sna) SEQ ID NO: 18 SEQ ID NO: 22

Example 2. Production of Corynebacterium glutamicum microorganisms introduced with anion transport proteins derived from foreign microorganisms

After synthesizing each nucleic acid (Table 1) encoding anion transport proteins of the obtained microorganisms, PCR was performed using each of these nucleic acids as a template and using the primer sequences in Table 1 above. PCR was performed using PfuUltraTM high-confidence DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95°C for 30 seconds; Annealing 55°C, 30 seconds; And the polymerization reaction was repeated 30 times at 72°C for 2 minutes. As a result, DNA fragments encoding each anion transport protein were obtained. To secure the lysC promoter derived from Corynebacterium Glutamicum, primers of SEQ ID NO: 11 and SEQ ID NO: 12 were used using Corynebacterium glutamicum genomic DNA as a template, and the promoter was used in the same manner as above. PCR was performed to obtain DNA fragments. pECCG117 (Korean Patent No. 10-0057684) vector treated with restriction enzyme ScaI and then heat-treated at 65°C for 20 minutes and the above DNA fragments (each Transporter, PlysC) were mixed at a molar concentration (M) of 2:1:1. A plasmid was obtained by cloning according to the provided manual using TaKaRa's Infusion Cloning Kit, and the name of the vector and information on the introduced gene are indicated in Table 1 above.

Five types of vectors were transformed into Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC) (European Patent Publication EP 4032977 A1) by electroporation to produce microorganisms expressing foreign anion transport proteins. did.

Example 3. Investigation of the pantothenic acid production ability of Corynebacterium glutamicum microorganisms expressing anion transport proteins derived from foreign microorganisms

In order to confirm the pantothenic acid production ability of the strains obtained in Example 2, the parent strain and the above strains were each inoculated into a 250 ml corner-bottle flask containing 25 ml of the production medium composed of the following composition, and then incubated at 32°C for 48 hours. Pantothenic acid was prepared by shaking culture at 200 rpm for 1 hour.

<Production Badge>

Glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, monobasic potassium phosphate 0.1%, magnesium sulfate 7-hydrate, 0.05%, iron sulfate 7-hydrate, 10 mg/ℓ, manganese sulfate mono-hydrate 6.7 mg/ℓ , biotin 50 ㎍/ℓ, thiamine·HCl 100 ㎍/ℓ, pH 7.2

The pantothenic acid production capacity of Corynebacterium glutamicum, which expresses anion transport proteins derived from various microorganisms, is summarized in Table 2 below.

strain Pantothenic acid (g/L) ATCC13032 ΔpanB::panB(EC) (parent strain) 0.28 ATCC13032 ΔpanB::panB(EC) pECCG117-Ncam0154 0.69 ATCC13032 ΔpanB::panB(EC) pECCG117-Transporter(Bsa) 0.29 ATCC13032 ΔpanB::panB(EC) pECCG117-Transporter(Kar) 0.30 ATCC13032 ΔpanB::panB(EC) pECCG117-Transporter(Rop) 0.26 ATCC13032 ΔpanB::panB(EC) pECCG117-Transporter(Sna) 0.23

As shown in Table 2, it was confirmed that the parent strain, Corynebacterium glutamicum ATCC13032 ΔpanB::panB(EC), produced about 0.28 g/L of pantothenic acid. Among strains expressing anion transport proteins derived from foreign microorganisms, ATCC13032 ΔpanB::panB(EC) pECCG117-Ncam0154, a strain expressing the anion transport protein Ncam0154 (SEQ ID NO. 14) derived from Corynebacterium stasis, weighed 0.69 g. /L of pantothenic acid was produced, showing the highest pantothenic acid productivity.

The above results mean that among the five types of anion transport proteins selected in this application, Ncam0154, an anion transport protein derived from Corynebacterium stasis, can produce pantothenic acid more efficiently, and that the anion transport protein is capable of producing pantothenic acid more efficiently. It was confirmed that production was increased.

Example 4. Investigation of pantothenic acid production ability of wild-type Corynebacterium glutamicum microorganism with enhanced Ncam0154 protein activity

From the results of Example 3, it was confirmed whether the Ncam0154 protein, which showed the best pantothenic acid productivity, showed a similar effect of improving pantothenic acid production ability when introduced into wild-type Corynebacterium glutamicum.

Specifically, the pECCG117-Ncam0154 vector constructed in Example 2 was transformed into Corynebacterium glutamicum ATCC13032 by electroporation to produce a strain expressing Ncam0154 (ATCC13032 pECCG117-Ncam0154). The prepared strains were inoculated together with the parent strain (ATCC13932) into a 250 ml corner-barpool flask containing 25 ml of production medium composed of the following composition, and then cultured with shaking at 200 rpm at 32°C for 48 hours, followed by pantothenic acid. Productivity was measured.

<Production Badge>

Glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, monobasic potassium phosphate 0.1%, magnesium sulfate 7-hydrate, 0.05%, iron sulfate 7-hydrate, 10 mg/ℓ, manganese sulfate mono-hydrate 6.7 mg/ℓ , biotin 50 ㎍/ℓ, thiamine·HCl 100 ㎍/ℓ, pH 7.2

strain Pantothenic acid (g/L) ATCC13032 0.08 ATCC13032pECCG117-Ncam0154 0.12

As a result, as shown in Table 3 above, it was confirmed that the Ncam0154 protein could improve the pantothenic acid production ability by 50% compared to the parent strain even when expressed in wild-type Corynebacterium glutamicum microorganisms.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Sequence list electronic file attached

Claims (13)

Microorganisms that produce pantothenic acid or pantoic acid with enhanced anion transport protein activity. The microorganism of claim 1, wherein the anion transport protein is from Corynebacterium stasis. The microorganism according to claim 1, wherein the anion transport protein is the Ncam0154 protein. The microorganism according to claim 1, wherein the anion transport protein comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity thereto. The microorganism according to claim 1, wherein the anion transport protein is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 13. The microorganism according to claim 1, wherein the microorganism has additionally enhanced activity of 3-methyl-2-oxobutanoate hydroxy methyltransferase. The microorganism according to claim 6, wherein the 3-methyl-2-oxobutanoate hydroxy methyltransferase is derived from Escherichia coli. The microorganism according to claim 1, wherein the microorganism is a microorganism of the genus Corynebacterium. The microorganism of claim 8, wherein the microorganism is Corynebacterium glutamicum. The microorganism according to any one of claims 1 to 9, wherein the microorganism has an increased ability to produce pantothenic acid or pantoic acid compared to the parent strain in which the activity of the anion transport protein is not enhanced. A method for producing pantothenic acid or pantoic acid, comprising culturing the microorganism of any one of claims 1 to 9 in a medium. The production method of claim 11, further comprising recovering pantothenic acid or pantoic acid from the culture medium or microorganism. A composition for producing pantothenic acid or pantoic acid, comprising the microorganism of any one of claims 1 to 9.