MICROORGANISM HAVING INCREASED ACTIVITY OF 3-METHYL-2-OXOBUTANOATE HYDROXYMETHYLTRANSFERASE, AND USE THEREOF

Abstract

Provided are: a polypeptide having activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase; a microorganism having increased activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase; a composition for producing pantothenic acid and/or pantoic acid, comprising the polypeptide and/or the microorganism, and a pantothenic acid and/or pantoic acid production method comprising a step for culturing the microorganism.

Claims

1. A microorganism of genus of Corynebacterium producing a pantothenic acid or pantoic acid, which has an enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase activity, wherein the 3-methyl-2-oxobutanoate hydroxymethyltransferase is derived from Escherichia Coli.

2. The microorganism of claim 1, wherein the 3-methyl-2-oxobutanoate hydroxymethyltransferase is PanB protein.

3. The microorganism of claim 1, wherein the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises the amino acid sequence of SEQ ID NO: 37.

4. The microorganism of claim 1, wherein the genus of Corynebacterium is Corynebacterium glutamicum.

5. A method of producing a pantothenic acid or pantoic acid, comprising: culturing the microorganism of claim 1 in a medium.

6. The method of producing pantothenic acid or pantoic acid of claim 5, further comprising, after the step of culturing, recovering the pantothenic acid or pantoic acid from the cultured microorganism, the medium, or both of them.

Description

MODE FOR INVENTION

[0058] Hereinafter, the present disclosure will be described in more detail with examples, but these examples are only for illustrative purpose and are not intended to limit the scope of the disclosure. It is clear to a person skilled in the art that the examples described below may be modified without departing from the spirit of the disclosure.

Example 1) Screening and Selection of 3-methyl-2-oxobutanoate Hydroxymethyltransferase Genes

[0059] As a result of conducting NCBI BLAST searching using the 3-methyl-2-oxobutanoate hydroxymethyltransferase coding gene (panB) of Corynebacterium glutamicum ATCC13032 as a query, candidate genes, which are estimated as having a 3-methyl-2-oxobutanoate hydroxymethyltransferase coding gene activity, and microorganisms comprising the candidate gene were selected. Among the microorganisms, 3-methyl-2-oxobutanoate hydroxymethyltransferase coding genes, which are derived from microorganisms having biosafety level 1 among the microorganisms, were selected and summarized in Table 1:

TABLE-US-00001 TABLE 1 Microorganisms expected as comprising 3-methyl-2-oxobutanoate hydroxymethyltransferase coding gene and primers/plasmids used in selecting the 3-methyl-2-oxobutanoate hydroxymethyltransferase genes Microorganism name KCTC Accession No. primers plasmid 1 Escherichia coli ATCC47076 SEQ ID NO: 1, 2 pECCG117-panB(EC) 2 Bacillus subtilis KCTC3135(ATCC 6051) SEQ ID NO: 3, 4 pECCG117-panB(BS) 3 Pantoea agglomerans KCTC2564(ATCC27155) SEQ ID NO: 5, 6 pECCG117-panB(PA) 4 Serratia rubidaea KCTC2927(ATCC27593) SEQ ID NO: 7, 8 pECCG117-panB(SR) 5 Serratia proteamaculans KCTC2936(ATCC19323) SEQ ID NO: 9, 10 pECCG117-panB(SP) 6 Pseudomonas resinovorans KCTC12498(ATCC14235) SEQ ID NO: 11, 12 pECCG117-panB(PR) 7 Pedobacter terrae KCTC12762(DSM17933) SEQ ID NO: 13, 14 pECCG117-panB(PT) 8 Citrobacter bitternis KCTC42139(JCM30009) SEQ ID NO: 15, 16 pECCG117-panB(CB) 9 Enterobacter cloacae KCTC2519(ATCC23355) SEQ ID NO: 17, 18 pECCG117-panB(ECI) 10 Achromobacter piechaudii KCTC22890(ATCC43552) SEQ ID NO: 19, 20 pECCG117-panB(AP) 11 Staphylococcus epidermidis KCTC1917(ATCC12228) SEQ ID NO: 21, 22 pECCG117-panB(SE) 12 Shigella flexneri KCTC12073 SEQ ID NO: 23, 24 pECCG117-panB(SF) 13 Corynebacterium glutamicum KCTC9097(ATCC13032) SEQ ID NO: 25, 26 pECCG117-panB(CG)

Example 2) Preparation of Microorganism Belonging to Genus Corynebacterium to Which Foreign Microorganism Derived 3-methyl-2-oxobutanoate Hydroxymethyltransferase is Introduced

[0060] After extracting genomes from the microorganisms obtained in Example 1, a PCR was performed using the genomes as templates and using the primers of Table 1, to amplify DNA fragments encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase. The PCR was conducted using PfuUltra high-fidelity DNA polymerase (Stratagene), by repeating 30 cycles of denaturing at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerizing at 72 C. for 1 minute. As the results, each 3-methyl-2-oxobutanoate hydroxymethyltransferase coding DNA fragment (panB) was obtained.

[0061] To prepare Corynebacterium glutamicum derived PLM1 promoter, a PCR was performed using the genome DNA of Corynebacterium glutamicum (ATCC13032) as a template and using primers of SEQ ID NOS: 27 and 28 under the above conditions, to obtain the promoter DNA fragment.

[0062] pECCG117 vector (Korean Patent No. 10-0057684), which was treated with restriction enzyme BamHI followed by heat treatment at 65 C. for 20 minutes, and the above obtained DNA fragments (each panB, PLM1 promoter) were mixed in a ratio of 2:1:1 (pECCG117 vector: panB: PLM1) based on a molar concentration (M), and cloned using Infusion Cloning Kit (TakaRa) according to manufacturer's manual, to obtain plasmids. Names of the obtained plasmids and gene information which was introduced thereto were summarized in Table 1.

[0063] The 13 obtained vectors were transformed into Corynebacterium glutamicum ATCC 13032 by electroporation, thereby preparing strains expressing exogenous PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase).

Example 3) Examination of Pantothenic Acid Production Ability of Foreign Microorganism-Derived 3-methyl-2-oxobutanoate Hydroxymethyltransferase Expressing Microorganism Belonging to Genus Corynebacterium

[0064] In order to examine pantothenic acid productivity of the strains expressing various foreign microorganism-derived panB prepared in Example 2, the strains and a parent strain (non-transformed strain) were respectively inoculated into a 250 ml corner-baffle flask containing 25 ml of a production medium, and then, cultured at 32 C. for 48 hours with shaking at 200 rpm, to produce pantothenic acid.

Production Medium

[0065] glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 10 mg/L, manganese sulfate monohydrate 6.7 mg/L, biotin 50 g/L, thiamine.Math.HCl 100 g/L, pH 7.2

[0066] The obtained culture solution was centrifuged at 20,000rcf for 10 minutes, and then, the supernatant liquid was diluted to 1/10 with TDW (triple distilled water) and subjected to HPLC analysis to measure the concentrations of pantothenic acid and L-valine. The obtained results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 pantothenic acid L-valine concentration concentration (g/L) (g/L) ATCC13032 (wild-type) 0.0 2.4 ATCC13032 pECCG117-panB(EC) 1.2 1.5 ATCC13032 pECCG117-panB(BS) 0.5 1.9 ATCC13032 pECCG117-panB(PA) 0.6 2.0 ATCC13032 pECCG117-panB(SR) 0.7 1.8 ATCC13032 pECCG117-panB(SP) 0.3 2.2 ATCC13032 pECCG117-panB(PR) 0.7 1.9 ATCC13032 pECCG117-panB(PT) 0.7 2.0 ATCC13032 pECCG117-panB(CB) 0.7 2.0 ATCC13032 pECCG117-panB(ECI) 0.6 2.1 ATCC13032 pECCG117-panB(AP) 0.6 1.9 ATCC13032 pECCG117-panB(SE) 0.5 2.0 ATCC13032 pECCG117-panB(SF) 0.7 1.9 ATCC13032 pECCG117-panB(CG) 0.6 2.0

[0067] As shown in Table 2, the parent strain, Corynebacterium glutamicum ATCC 13032, does not produce pantothenic acid, whereas all the tested foreign microorganism-derived panB expressing Corynebacterium glutamicum strains produced pantothenic acid of about 0.6 g/L in average. In particular, among the foreign microorganism-derived panB expressing strains, foreign E. coli-derived PanB expressing strain, ATCC13032 pECCG117-panB (EC), exhibited the highest pantothenic acid productivity (1.2 g/L).

[0068] The above results show that all 13 microorganism-derived enzymes (3-methyl-2-oxobutanoate hydroxymethyltransferases) selected in Example 1 exhibit pantothenic acid production ability, and among them, E. coli-derived enzyme hasspecially high pantothenic acid production ability.

[0069] Example 4) Preparation of Microorganism Belonging to Genus Corynebacterium to which E. coli-Ferived 3-methyl-2-oxobutanoate Hydroxymethyltransferase Gene is Introduced

[0070] A plasmid was prepared for introducing an E. coli-derived 3-methyl-2-oxobutanoate hydroxymethyltransferase coding gene (panB), which was evidenced as having excellent pantothenic acid production ability in Example 3, into Corynebacterium glutamicum ATCC13032.

[0071] First, a vector for deleting panB present in the parent strain (wild-type) was constructed. A PCR was performed using the genome DNA of Corynebacterium glutamicum ATCC13032 as a template and using the primers of SEQ ID NOS: 29 and 30, and SEQ ID NOS: 31 and 32. The PCR was performed by repeating 25 cycles of denaturing at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerization at 72 C. for 1 minute. As a result, a gene fragment of 1000 bp at upstream region of the panB gene and a gene fragment of 1000 bp at downstream region of the panB gene were obtained, respectively. Each amplification product was purified using QIAGEN's PCR purification kit and used as an insert DNA fragment for vector construction.

[0072] pDZ vector (U.S. Pat. No. 9,109,242 B2), which was treated with restriction enzyme smal followed by heat treatment at 65 C. for 20 minutes, and the DNA fragments (the gene fragment of 1000 bp at upstream region of the panB gene and a gene fragment of 1000 bp at downstream region of the panB gene panB) were mixed in a ratio of 2:1:1 based on a molar concentration (M), and cloned using Infusion Cloning Kit (TakaRa) according to manufacturer's manual, to construct pDZ_panB vector for deleting the panB gene from the chromosome.

[0073] To provide an E. coli-derived panB gene, a PCR was performed using plasmid pECCG117-panB (EC) prepared in Example 2 as a template and using primers of SEQ ID NOS: 33 and 34. The PCR was performed by repeating 25 cycles of denaturing at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerization at 72 C. for 1 minute. As a result, a DNA fragment of 1077 bp was obtained. The pDZ_panB vector, which was treated with restriction enzyme smal followed by heat treatment at 65 C. for 20 minutes, and the above obtained DNA fragment, were mixed in a ratio of 1:2 based on a molar concentration (M), and cloned using Infusion Cloning Kit (TakaRa) according to manufacturer's manual, to construct pDZ_panB::PLM1-panB (EC) vector for introducing the E. coli-derived panB gene into the chromosome.

[0074] Corynebacterium glutamicum ATCC 13032 was transformed with each of the constructed vectors pDZ_panB and pDZ_panB::panB (EC), and then subjected to a secondary crossover, thereby preparing a strain (panB strain) in which panB was deleted from its chromosome and a strain (panB::panB (EC) strain) in which panB was deleted from its chromosome and the E. coli-derived panB gene was introduced into the chromosome, respectively. A proper substitution with E. coli-derived panB gene was confirmed by MASA (Mutant Allele Specific Amplification) PCR method (Takeda et al., Hum. Mutation, 2, 112-117 (1993)) using primer combinations as below. Than is, a first determination was made by selecting strains that were amplified using a primer combination suitable for E. coli, SEQ ID NOS: 35 and 28, and SEQ ID NOS: 36 and 1, and then, a second conformation was made by analyzing the sequence of panB gene of the selected strains using a primer combination of SEQ ID NO: 35 and SEQ ID NO: 36.

[0075] To examine pantothenic acid production abilities of the above obtained mutants, Corynebacterium glutamicum ATCC 13032 wild-type strains, panB strains, and panB::panB (EC) mutants were respectively inoculated into 250 ml corner-baffle flask containing 25 ml of a production medium (referring to Example 3), and then, cultured at 32 C. for 48 hours with shaking at 200 rpm, to produce pantothenic acid.

[0076] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then, the supernatant liquid was diluted to 1/10 with TDW (triple distilled water) and subjected to HPLC analysis to measure the concentrations of pantothenic acid and L-valine. The obtained results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 pantothenic acid L-valine concentration concentration (g/L) (g/L) ATCC13032 (wild-type) 0.1 1.9 ATCC13032 panB 0.0 2.7 ATCC13032 panB ::panB(EC) 0.4 1.3

[0077] As shown in Table 3, wild-type Corynebacterium glutamicum ATCC 13032 and the panB deleted (panB) strain displayed no or very poor production of pantothenic acid, whereas the foreign panB-expressing mutant Corynebacterium glutamicum (panB::panB (EC) strain) produced pantothenic acid at the concentration of 0.4 g/L.

Example 5) Preparation of Randomly Mutated Strains by Artificial Mutation (NTG-Based Mutation) and Selection of panB-Producing Strains

[0078] In this example, in order to obtain microorganism mutants with more enhanced pantothenic acid production ability, a mutation of microorganism was induced as follows.

[0079] More specifically, Corynebacterium glutamicum ATCC panB::panB (EC) strains were activated by being cultured in an activation medium for 16 hours, inoculated on a seed medium sterilized at 121 C. for 15 minutes and cultured for 14 hours, and then, 5ml of the obtained culture solution was collected. The collected culture solution was washed with 100 mM citric acid buffer, NTG (N-Methyl-N-nitro-N-nitrosoguanidine) was added thereto so that its final concentration reaches to 200 mg/L and left for 20 minutes, and then, the resulted product was washed with 100 mM phosphate buffer. The NTG-treated strains were smeared on minimal medium, and their death rate was measured as 85%. The survived cells were inoculated and cultured on a production medium, and finally, a mutant exhibiting excellent pantothenic acid productivity were selected and named as Corynebacterium glutamicum CJVB5-01.

[0080] The compositions of the media used in this example were as follows:

Activation Medium

[0081] beef extract 1%, polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2

Seed Medium

[0082] glucose 5%, Bacto peptone 1%, sodium chloride 0.25%, yeast extract 1%, urea 0.4%, pH 7.2

Production Medium

[0083] glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 10 mg/L, manganese sulfate monohydrate 6.7 mg/L, biotin 50 g/L, thiamine.Math.HCl 100 g/L, pH 7.2

Minimal Medium

[0084] glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, monopotassium phosphate 0.1%, urea 0.1%, thiamine 0.001%, biotin 200 g/L, agar 2%, pH 7.2

[0085] To examine pantothenic acid production ability of the above obtained mutant Corynebacterium glutamicum CJVB5-01, Corynebacterium glutamicum panB strain, panB: panB (EC) strain, and CJVB5-01 mutant were respectively inoculated into 250 ml corner-baffle flask containing 25 ml of a production medium, and then, cultured at 32 C. for 48 hours with shaking at 200 rpm, to produce pantothenic acid.

[0086] The obtained culture solution was centrifuged at 20,000rcf for 10 minutes, and then, the supernatant liquid was diluted to 1/10 with TDW (triple distilled water) and subjected to HPLC analysis to measure the concentrations of pantothenic acid and L-valine. The obtained results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Pantothenic acid productivity of NTG-based mutated strains pantothenic acid L-valine concentration concentration (g/L) (g/L) ATCC13032 panB 0.0 2.4 ATCC13032 panB ::panB(EC) 0.3 1.4 CJVB5-01 1.2 1.0

[0087] As shown in Table 4, Corynebacterium glutamicum panB did not produce pantothenic acid, whereas foreign panB-inserted Corynebacterium glutamicum panB::panB (EC) strain produced pantothenic acid at the concentration of 0.3 g/L and the Corynebacterium glutamicum CJVB5-01 mutant produced pantothenic acid at the concentration of 1.2 g/L. From these results, it was confirmed that Corynebacterium glutamicum CJVB5-01 mutant displays more excellent pantothenic acid productivity.

[0088] From the genome sequencing result of the Corynebacterium glutamicum CJVB5-01 mutant, it was confirmed that the inserted E. coli panB gene is mutated, so as to encode a variant of wild-type E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase (SEQ ID NO: 37), to which G116A mutation (substitution of the amino acid corresponding to position 116 of the amino acid sequence of SEQ ID NO: 37, G (Gly), with A (Ala)) is introduced. Hereinafter, the indication of an amino acid mutation using amino acid position, such as G116A, may be understood to mean an amino acid mutation and/or a genetic mutation leading to such amino acid mutation.

[0089] The amino acid sequence of the E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase variant, to which G116A mutation is introduced, was indicated as SEQ ID NO: 62.

[0090] From the results, it was confirmed that the mutant obtained through the random mutagenesis method can produce pantothenic acid with high efficiency and high yield without inhibiting the pathway for pantothenic acid synthesis from pyruvic acid.

Example 6) Preparation of Mutant PanB Plasmid Having 3-methyl-2-oxobutanoate Hydroxymethyltransferase Activity

[0091] In order to examine that the amino acid residue at position 116, which is a mutation position of the E. coli PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase) and confirmed as affecting pantothenic acid productivity through Example 5, is important in increasing the pantothenic acid productivity, variants in which the position is substitution with various other amino acids were prepared and the effects thereof were examined. Using pECCG117-panB (EC) (referring to Table 1) prepared in Example 2 as a template and using primers of Table 5 below, 19 variants having a random mutation (saturated mutagenesis) in which the amino acid of position 116 of E. coli PanB (SEQ ID NO: 37), G (Gly), is substituted with other amino acid (that is, the variants are mutated by introducing mutated panB gene encoding the randomly mutated E. coli PanB) were prepared. The substituted amino acids of the variant mutated by each saturated mutagenesis and primers used therefor are summarized in Table 5 below:

TABLE-US-00005 TABLE 5 Template Amino acid substitution Primers used pECCG117- G116S SEQ ID NO: 27, 38/39, 28 panB(EC) G116C SEQ ID NO: 27, 40/41, 28 G116L SEQ ID NO: 27, 42/43, 28 G116I SEQ ID NO: 27, 44/45, 28 G116T SEQ ID NO: 27, 46/47, 28 G116V SEQ ID NO: 27, 48/49, 28 G116M SEQ ID NO: 27, 50/51, 28 G116D SEQ ID NO: 27, 52/53, 28 G116E SEQ ID NO: 27, 54/55, 28 G116N SEQ ID NO: 27, 56/57, 28 G116Q SEQ ID NO: 27, 58/59, 28 G116A SEQ ID NO: 27, 60/61, 28

[0092] In particular, a PCR was performed using primers presented in Table 5 and using pECCG117-panB (EC) (Table 1) prepared in Example 2 as a template. As a polymerase, Solg Pfu-X DNA polymerase (SolGent co., Ltd.) was employed. The PCR was performed by repeating 25 cycles of denaturing at 95 C. for 10 minutes, followed by denaturing at 95 C. for 30 seconds, annealing at 55 C. for 30 seconds, and polymerizing at 72 C. for 1 minute. As a result, DNA fragments of 610bp of 5 upstream region and 470 bp of 3 downstream region centering the mutation site of 3-methyl-2-oxobutanoate hydroxymethyltransferase gene were obtained.

[0093] pECCG117 vector (Korean Patent No. 10-0057684), which was treated with restriction enzyme BamHI followed by heat treatment at 65 C. for 20 minutes, and the above obtained DNA fragments (5 upstream 610bp DNA fragment and 3 downstream 470 bp DNA fragment) were mixed in a ratio of 2:1:1 based on a molar concentration (M), and cloned using Infusion Cloning Kit (TakaRa) according to manufacturer's manual, to obtain 19 mutant plasmids for introducing mutated panB. The information of the 19 mutant plasmids was summarized in Table 6.

TABLE-US-00006 TABLE 6 Mutation Amino acid Mutant plasmid prepared for inducing position substitution the amino acid substitution amino acid G116S pECCG117-panB(G116S) residue at G116C pECCG117-panB(G116C) position 116 G116L pECCG117-panB(G116L) of PanB G116I pECCG117-panB(G116I) (SEQ ID G116T pECCG117-panB(G116T) NO: 37) G116V pECCG117-panB(G116V) G116M pECCG117-panB(G116M) G116D pECCG117-panB(G116D) G116E pECCG117-panB(G116E) G116N pECCG117-panB(G116N) G116Q pECCG117-panB(G116Q) G116A pECCG117-panB(G116A)

Example 7) Measurement of Pantothenic Acid Productivity of panB Variants Having muta3-methyl-2-oxobutanoate Hydroxymethyltransferase Activity

[0094] The mutant plasmid prepared in Example 6 and pECCG117-panB (WT-EC) (Table 1) were respectively introduced into the ATCC13032 panB strain prepared in Example 4 by electric pulse method, and then, smeared on a selective medium containing 25 mg/L of kanamycin, to obtain a total of 19 transformed mutant strains to which each random mutation (saturated mutagenesis) was introduced. Thereafter, a flask test was performed in the same manner as in Example 3, and pantothenic acid production abilities of the transformed mutant strains were measured. The results are shown in Table 7:

TABLE-US-00007 TABLE 7 pantothenic acid (g/L) Strain Batch 1 Batch 2 Batch 3 Average ATCC 13032 panB (control) 0.0 0.0 0.0 0.0 ATCC 13032 panB pECCG117- 1.5 1.9 1.6 1.7 panB(G116S) ATCC 13032 panB pECCG117- 1.2 1.3 1.2 1.2 panB(G116C) ATCC 13032 panB pECCG117- 1.6 1.5 1.4 1.5 panB(G116L) ATCC 13032 panB pECCG117- 1.5 1.7 1.6 1.6 panB(G116I) ATCC 13032 panB pECCG117- 2.4 2.5 2.3 2.4 panB(G116T) ATCC 13032 panB pECCG117- 1.6 1.7 1.4 1.6 panB(G116V) ATCC 13032 panB pECCG117- 0.9 0.9 1.1 1.0 panB(G116M) ATCC 13032 panB pECCG117- 1.3 1.1 1.2 1.2 panB(G116D) ATCC 13032 panB pECCG117- 1.9 1.2 2.1 1.7 panB(G116E) ATCC 13032 panB pECCG117- 2.5 2.5 2.6 2.5 panB(G116N) ATCC 13032 panB pECCG117- 1.0 1.1 1.0 1.0 panB(G116Q) ATCC 13032 panB pECCG117- 2.6 2.9 2.9 2.8 panB(G116A) ATCC 13032 panB pECCG117- 0.9 0.8 1.0 0.9 panB(WT)

[0095] As shown in Table 7, ATCC13032 panB strain did not produce pantothenic acid, whereas all the mutant strains to which E. coli PanB (wild-type) or a variant thereof is introduced displayed pantothenic acid production ability. In addition, the mutant strain to which mutation G116S, G116C, G116L, G1161, G116T, G116V, G116D, G116E, G116N, G116A, G116M, or G116Q is introduced produced pantothenic acid at higher level compared with ATCC 13032 panB pECCG117-panB (WT) which is a mutant strain having E. coli PanB (wild-type). As a result, it was confirmed that both wild-type and mutant forms of E. coli PanB exhibit the effect of increasing pantothenic acid production, in particular, the amino acid residue at position 116 of PanB (SEQ ID NO: 37) is an important position in pantothenic acid production, and when the amino acid at this position is substituted with various amino acids different from the original, the production ability of pantothenic acid was further increased.

[0096] ATCC 13032 panB pECCG117-panB (G116A) strain (called as Corynebacterium glutamicum CV03-5001), which is confirmed as having the most excellent pantothenic acid producing ability in this example, was deposited with the Korea Culture Center of Microorganisms located in Hongje-dong, Seodaemun-gu, Seoul, Korea, a depository institution under the Budapest Treaty, on Jun. 8, 2020, and given the accession number KCCM12744P.

[0097] From the above description, it will be understood by those skilled in the art that the present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present application is to be interpreted as being within the scope of the present application, all changes or modifications derived from the meaning and scope of the appended Claim s and from their equivalents rather than the detailed description.