MICROORGANISM HAVING ENHANCED ACTIVITY OF 3-METHYL-2-OXOBUTANOATE HYDROXYMETHYLTRANSFERASE AND USES THEREOF

Abstract

A 3-methyl-2-oxobutanoate hydroxymethyltransferase variant, a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, a composition for producing pantothenic acid and/or pantoic acid comprising the microorganism, and a method for preparing pantothenic acid and/or pantoic acid comprising culturing the microorganism are provided.

Claims

1. A polypeptide, in which an 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.

2. The polypeptide according to claim 1, wherein the amino acid corresponding to the 159th residue is substituted with 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).

3. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of any one SED ID NO selected from the group consisting of amino acid sequences of SEQ ID NO: 110 to SEQ ID NO: 125.

4. The polypeptide according to claim 1, in which an amino acid corresponding to the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is further substituted with another amino acid.

5. The polypeptide according to claim 4, wherein the amino acid corresponding to the 116th residue from the N-terminus is 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).

6. The polypeptide according to claim 4, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 128.

7. A polynucleotide encoding the polypeptide of claim 1.

8. (canceled)

9. A microorganism producing pantothenic acid or pantoic acid, wherein the microorganism comprises the polypeptide according to claim 1, or a polynucleotide encoding the polypeptide.

10-13. (canceled)

14. The microorganism according to claim 9, wherein the microorganism is a genus Corynebacterium microorganism or a genus Escherichia microorganism.

15. The microorganism according to claim 14, wherein the genus Corynebacterium microorganism is Corynebacterium glutamicum.

16. (canceled)

17. A method for preparation of pantothenic acid or pantoic acid, comprising culturing the microorganism of claim 9 in a medium.

18. The method for preparation of pantothenic acid or pantoic acid of claim 17, further comprising recovering pantothenic acid or pantoic acid from the cultured microorganism, medium or both of them, after the culturing.

19. A polynucleotide encoding the polypeptide of claim 4.

20. A microorganism producing pantothenic acid or pantoic acid, wherein the microorganism comprises the polypeptide according to claim 4, or a polynucleotide encoding the polypeptide.

Description

MODE FOR INVENTION

[0076] Hereinafter, the present invention will be more specifically described by examples, but they are illustrative only, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the examples described below may be modified without departing from the essential gist of the invention.

Example 1. 3-Methyl-2-oxobutanoate Hydroxymethyltransferase Gene Search and Selection

[0077] As the result of NCBI BLAST search by setting a gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) of Corynebacterium glutamicum ATCC13032 to a query, candidate genes estimated as having activity of the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase and microorganisms having them were selected. Among them, the genes encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase derived from microorganisms having a biosafety level of 1 were selected, and they are summarized in Table 1:

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

Example 2. Production of the Genus Corynebacterium Microorganism in which Foreign Microorganism-Derived 3-Methyl-2-Oxobutanoate Hydroxymethyltransferase

[0078] After extracting genome of the microorganisms secured in Example 1, PCR was performed using the primer sequences of Table 1 using it as a template, and DNA fragments encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase were amplified. The PCR was performed using PfuUltra high-confidence DNA polymerase (Stratagene), and was performed under the condition of repeating denaturation at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerization at 72 C. for 1 minute 30 times. As a result, DNA fragments encoding each 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) were obtained.

[0079] In order to secure Corynebacterium glutamicum-derived PLM1 promoter, PCR was performed using primers of SEQ ID NOs: 27 and 28 using Corynebacterium glutamicum (ATCC13032) genome as a template as same as described above to obtain promoter DNA fragments.

[0080] After treating with restriction enzyme BamHI, by cloning pECCG117 vector heat-treated at 65 C. for 20 minutes (Korean Patent No. 10-0057684) and obtained DNA fragments (each panB, PLM1 promoter) to be a molarity (M) of 2:1:1 (pECCG117 vector: panB: PLM1) according to the provided manual using Infusion Cloning Kit of TakaRa, plasmids were obtained, and the names of the obtained plasmids and introduced gene information were marked in Table 1 above.

[0081] By transforming the produced 13 kinds of vectors into Corynebacterium glutamicum ATCC13032 by electroporation, strains expressing foreign PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase) were produced.

Example 3. Investigation of Pantothenic Acid Production Ability of the Genus Corynebacterium Microorganism in which Foreign Microorganism-Derived 3-Methyl-2-Oxobutanoate Hydroxymethyltransferase is Expressed

[0082] In order to confirm the productivity of pantothenic acid of various foreign microorganism-derived panB expressing strains obtained in Example 2, a parent strain (non-transformed strain) and the strains were inoculated into a 250 ml corner-baffle flask containing production medium 25 ml consisting of the following composition, respectively, and then cultured with shaking at 200 rpm at 32 C. for 48 hours to prepare pantothenic acid.

<Production Medium>

[0083] Glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium phosphate monobasic 0.1%, magnesium sulfate 7 hydrate 0.05%, iron sulfate 7 hydrate 10 mg/l, manganese sulfate 1 hydrate 6.7 mg/l, biotin 50 g/l, thiamine.Math.HCl 100 g/l, pH 7.2

[0084] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then the supernatant was diluted by 1/10 with TDW (triple distilled water), and then HPLC analysis was performed to measure the concentration of pantothenic acid and L-valine, and the result was 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(ECl) 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

[0085] As shown in Table 2, the parent strain, Corynebacterium glutamicum ATCC 13032 did not produce pantothenic acid, whereas all the tested Corynebacterium glutamicum strains expressing foreign microorganism-derived panB produced about 0.6 g/L pantothenic acid. In particular, among the foreign microorganism-derived PanB expressing strains, E. coli-derived panB expressing strain, ATCC13032 pECCG117-panB (EC) showed the highest pantothenic acid productivity (1.2 g/L).

[0086] The above result showed that 13 kinds of microorganism-derived enzymes (3-methyl-2-oxobutanoate hydroxymethyltransferase) selected in Example 1 showed the pantothenic acid production ability, and among them, the E. coli-derived enzyme showed especially high pantothenic acid production ability.

Example 4. Production of the Genus Corynebacterium Microorganism in which E. Coli-Derived 3-Methyl-2-Oxobutanoate Hydroxymethyltransferase Gene is Introduced

[0087] A plasmid to introduce the E. coli-derived 3-methyl-2-oxobutanoate hydroxymethyltransferase-encoding gene (panB) determined as having the especially excellent pantothenic acid production ability in Example 3 into Corynebacterium glutamicum ATCC13032 was produced.

[0088] At first, a vector to delete panB present in the parent strain was produced. PCR was performed using primers of SEQ ID NOs: 29 and 30 and SEQ ID NOs: 31 and 32 using the genome DNA of Corynebacterium glutamicum ATCC13032 as a template. PCR was performed under the condition of repeating denaturation at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerization at 72 C. for 1 minute 25 times. As a result, gene fragments of 1000 bp at the top region of the panB gene and 1000 bp at the bottom region of the panB gene were obtained, respectively, and each amplified product was purified using PCR Purification kit of QIAGEN and was used as insertion DNA fragments for vector production.

[0089] After treating with restriction enzyme smaI, by cloning pDZ vector heat-treated at 65 C. for 20 minutes (Korean Patent No. 0924065) and DNA fragments (gene fragments of 1000 bp at the top region of panB gene and gene fragments of 1000 bp at the bottom region of panB gene) to be a molarity (M) of 2:1:1 according to the provided manual using Infusion Cloning Kit of TakaRa, a vector to delete panB gene on chromosome, pDZ_panB was produced.

[0090] In order to prepare E. coli-derived panB gene, PCR was performed using primers of SEQ ID NOs: 33 and 34 using the plasmid pECCG117-panB (EC) produced in Example 2 as a template. PCR was performed by repeating denaturation at 95 C. for 30 seconds; annealing at 55 C. for 30 seconds; and polymerization at 72 C. for 1 minute 25 times, and as a result, DNA fragments of 1077 bp were obtained. After treating with restriction enzyme smaI, by cloning pDZ_panB vector heat-treated at 65 C. for 20 minutes and the obtained DNA fragments to be a molarity (M) of 1:2 according to the provided manual using Infusion Cloning Kit of TakaRa, a vector to introduce E. coli-derived panB gene on chromosome, pDZ_panB::PLM1-panB (EC) was produced.

[0091] The produced vectors, pDZ_panB and pDZ_panB::panB (EC) were transformed into Corynebacterium glutamicum ATCC13032, respectively, by electroporation, and through a secondary cross process, a strain in which panB was deleted (panB strain) and a strain in which E. coli-derived panB was introduced (panB::panB (EC)) were obtained on chromosome, respectively. Whether or not the appropriate substitution of E. coli-derived panB was confirmed using the following primer combination using MASA (Mutant Allele Specific Amplification) PCR technique (Takeda et al., Hum. Mutation, 2, 112-117 (1993)). In other words, primary determination was made by selecting strains amplified in the primer combination corresponding to E. coli panB (SEQ ID NOs: 35 and 28 and SEQ ID NOs: 36 and 1), and secondary confirmation was conducted by analyzing the panB sequence of the selected strain using the primer combination of SEQ ID NO: 35 and SEQ ID NO: 36.

[0092] In order to confirm the pantothenic acid productivity of the mutant strains obtained as described above, after inoculating Corynebacterium glutamicum ATCC 13032 wild type strain, panB strain, and panB::panB (EC) mutant strain into a 250 ml corner-baffle flask containing production medium (see Example 3) 25 ml, they were cultured with shaking at 300 rpm at 32 C. for 48 hours to prepare pantothenic acid.

[0093] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then the supernatant was diluted by 1/10 with TDW (triple distilled water), and then HPLC analysis was performed to measure the concentration of pantothenic acid and L-valine, and the result was shown in Table 3 below.

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

[0094] As shown in Table 3, the Corynebacterium glutamicum ATCC 13032 wild type and panB deleted (panB) strain did not produce pantothenic acid at all or hardly produced it, whereas the mutant Corynebacterium glutamicum expressing foreign panB (panB::panB (EC)) produced pantothenic acid at a concentration of 0.4g/l.

Example 5. Random Mutant Strain Production Through Artificial Mutation (NTG-Based Mutation) and panB Producing Strain Selection

[0095] In the present example, in order to obtain a microorganism mutant strain with much more enhanced production ability of pantothenic acid, using the following method, a mutation of the microorganism was induced using the mutant Corynebacterium glutamicum ATCC13032 panB::panB (EC) strain expressing E. coli-derived panB produced according to Example 4 as a parent strain.

[0096] Specifically, the Corynebacterium glutamicum ATCC13032 panB::panB (EC) strain was activated by culturing it in an activation medium for 16 hours, and was inoculated in a seed medium sterilized at 121 C. for 15 minutes and cultured for 14 hours, and then the culture solution 5 ml was recovered. After washing the recovered culture solution with 100 mM citric buffer, NTG (N-Methyl-N-nitro-N-nitrosoguanidine) was added so as to be the final concentration of 200 mg/l and was treated for 20 minutes, and was washed with 100 mM phosphate buffer. As the result of measuring the death rate by smearing the NTG-treated strain to a minimal medium, the death rate was shown as 85%. The survived cells were inoculated and cultured, and finally, 2 kinds of mutant strains showing the excellent pantothenic acid production ability were selected and named Corynebacterium glutamicum CJVB5-01 and CJVB5-02 (Corynebacterium glutamicum, CJVB5-02).

[0097] The composition of the medium used in the present example was as follows.

<Activation Medium>

[0098] Beef extract 1%, polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2

<Production Medium>

[0099] Glucose 10%, beta-alanine 0.5%, yeast extract 0.4%, ammonium sulfate 1.5%, potassium phosphate monobasic 0.1%, magnesium sulfate 7 hydrate 0.05%, iron sulfate 7 hydrate 10 mg/l, manganese sulfate 1 hydrate 6.7 mg/l, biotin 50 g/l, thiamine.Math.HCl 100 g/l, pH 7.2

<Minimal Medium>

[0100] Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate monobasic 0.1%, urea 0.1%, thiamine 0.001%, biotin 200 g/l, agar 2%, pH 7.2

[0101] In order to confirm the pantothenic acid production ability of the obtained mutant strains Corynebacterium glutamicum CJVB5-01 and CJVB5-02, after inoculating Corynebacterium glutamicum ATCC13032 panB::panB (EC) strain, CJVB5-01 mutant strain and CJVB5-02 mutant strain into a 250 ml corner-baffle flask containing production medium 25 ml, respectively, they were cultured with shaking at 200 rpm at 32 C. for 48 hours to prepare pantothenic acid.

[0102] The obtained culture solution was centrifuged at 20,000 rcf for 10 minutes, and then the supernatant was diluted by 1/10 with TDW (triple distilled water), and then HPLC analysis was performed to measure the concentration of pantothenic acid and L-valine, and the result was shown in Table 4 and Table 5 below.

TABLE-US-00004 TABLE 4 Pantothenic acid L-valine concentration concentration (g/L) (g/L) ATCC13032 panB 0.0 2.4 ATCC13032 0.3 1.4 panB::panB(EC) CJVB5-01 1.2 1.0

TABLE-US-00005 TABLE 5 Pantothenic acid L-valine concentration (g/L) concentration (g/L) ATCC13032 0.3 0.9 panB::panB(EC) CJVB5-02 1.7 0.2

[0103] As shown in Table 4 and Table 5 above, it was confirmed that Corynebacterium glutamicum panB strain did not produce pantothenic acid, and the Corynebacterium glutamicum CJVB5-01 mutant strain and CJVB5-02 mutant strain showed more excellent pantothenic acid production ability, compared to the foreign panB-inserted Corynebacterium glutamicum panB::panB (EC). In addition, it could be confirmed that the pantothenic acid production ability of the CJVB5-1 mutant strain and CJVB5-02 mutant strain was much stronger than that of the wild type, considering that the concentration of valine, a substance using 3-methyl-2-oxobutanoate as a substrate decreased.

[0104] As the result of genome sequencing of the Corynebacterium glutamicum CJVB5-01 mutant strain, it was confirmed that the inserted E. coli panB gene was mutated to encode a variant in which G116A mutation (substitution of the 116th amino acid residue in the amino acid sequence of SEQ ID NO: 37, G (Gly) with A (Ala)) was introduced into wild type E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase (SEQ ID NO: 37). Hereinafter, the indication of amino acid mutations using amino acid positions such as G116A is understood to mean amino acid mutations and/or genetic mutations inducing such amino acid mutations. The amino acid sequence of of the E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase variant in which the G116A mutation was introduced was shown in SEQ ID NO: 62.

[0105] As the result of genome sequencing of the Corynebacterium glutamicum CJVB5-02 mutant strain, it was confirmed that the inserted E. coli panB gene was mutated to encode a variant in which A159L mutation (substitution of the 159th amino acid residue in the amino acid sequence of SEQ ID NO: 37, A (Ala, alanine) with L (Leu, leucine)) was introduced into wild type E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase (SEQ ID NO: 37). Hereinafter, the indication of amino acid mutations using amino acid positions such as A159L is understood to mean amino acid mutations and/or genetic mutations inducing such amino acid mutations. The amino acid sequence of the E. coli 3-methyl-2-oxobutanoate hydroxymethyltransferase variant in which the A159L mutation was introduced was shown in SEQ ID NO: 110.

[0106] As a result, it was confirmed that the CJVB5-01 mutant strain and CJVB5-02 mutant strain produced pantothenic acid with high efficiency and high yield, without inhibiting a pathway of synthesizing pantothenic acid from pyruvic acid.

Example 6. Production of Mutant panB Plasmid Having 3-Methyl-2Ketobutanoate Hydroxymethyltransferase Activity

[0107] In order to confirm that the amino acid residues corresponding to the 116th and/or 159th residues, positions of mutations of E. coli PanB (3-methyl-2-oxobutanoate hydroxymethyltransferase) confirmed as affecting the pantothenic acid production ability through Example 5 were important positions, a mutant in which amino acid residues at these positions were substituted with other amino acids was produced and the effect was confirmed.

[0108] Using primers described in Table 6 below using pECCG117-panB (EC) produced in Example 2 (See Table 1) as a template, a variant in which random mutagenesis (saturated mutagenesis) in which the amino acid at the 116th position of E. coli PanB (SEQ ID NO: 37), G (Gly) was substituted with another amino acid was introduced (that is, the panB gene mutated to encode E. coli PanB in which the random mutagenesis was introduced was introduced) was produced. In addition, using primers described in Table 7 below, a variant, in which random mutagenesis in which the amino acid at the 159th position of E. coli PanB (SEQ ID NO: 37), A (Ala) was substituted with another amino acid was introduced, was produced. The pECCG117-panB (EC) produced in Example 1 was used as a template. The amino acids substituted according to the mutant of the mutant strain in which each saturated mutagenesis was introduced and primers used for each mutant were summarized in Table 6 and Table 7 below:

TABLE-US-00006 TABLE 6 Amino acid Template substitution Used primer pECCG117-panB(EC) G116S SEQ ID NOs: 27, 38/39, 28 G116C SEQ ID NOs: 27, 40/41, 28 G116L SEQ ID NOs: 27, 42/43, 28 G116I SEQ ID NOs: 27, 44/45, 28 G116T SEQ ID NOs: 27, 46/47, 28 G116V SEQ ID NOs: 27, 48/49, 28 G116M SEQ ID NOs: 27, 50/51, 28 G116D SEQ ID NOs: 27, 52/53, 28 G116E SEQ ID NOs: 27, 54/55, 28 G116N SEQ ID NOs: 27, 56/57, 28 G116Q SEQ ID NOs: 27, 58/59, 28 G116A SEQ ID NOs: 27, 60/61, 28

TABLE-US-00007 TABLE 7 Substituted amino Template acid Used primer pECCG117- A159R SEQ ID NOs: 74, 75/76, 77 panB(EC) A159S SEQ ID NOs: 74, 78/79, 77 A159Y SEQ ID NOs: 74, 80/81, 77 A159C SEQ ID NOs: 74, 82/83, 77 A159P SEQ ID NOs: 74, 84/85, 77 A159H SEQ ID NOs: 74, 86/87, 77 A159L SEQ ID NOs: 74, 88/89, 77 A159I SEQ ID NOs: 74, 90/91, 77 A159T SEQ ID NOs: 74, 92/93, 77 A159K SEQ ID NOs: 74, 94/95, 77 A159V SEQ ID NOs: 74, 96/97, 77 A159M SEQ ID NOs: 74, 98/99, 77 A159D SEQ ID NOs: 74, 100/101, 77 A159E SEQ ID NOs: 74, 102/103, 77 A159N SEQ ID NOs: 74, 104/105, 77 A159Q SEQ ID NOs: 74, 106/107, 77 WT (SEQ ID NO: 37) SEQ ID NOs: 74, 77

[0109] Specifically, using primers presented in Table 6 and Table 7 above, PCR was performed using pECCG117-panB (EC) (See Table 1) produced in Example 2 as a template. As polymerase, Solg Pfu-X DNA polymerase (SolGent co., Ltd.) was used, and PCR was progressed by denaturing at 95 C. for 10 minutes, and then repeating denaturation at 95 C. for 30 seconds, annealing at 55 C. for 30 seconds and polymerization at 72 C. for 1 minute 25 times, and then performing polymerization at 72 C. for 5 minutes. As a result, 610 bp DNA fragments at the 5 upstream region and 470 bp DNA fragments at the 3 downstream region centering on the mutation (116th residue) of 3-methyl-2-oxobutanoate hydroxymethyltransferase gene were obtained, and 477 bp DNA fragments at the 5 upstream region and 318 bp DNA fragments at the 3 downstream region centering on the 159th residue were obtained.

[0110] After treating with restriction enzyme BamHI, by cloning pECCG117 vector heat-treated at 65 C. for 20 minutes (Korean Patent No. 10-0057684) and obtained each DNA fragment (116th residue: 610 bp DNA fragments at the 5 upstream region and 470 bp DNA fragments at the 3 downstream region, 159th residue: 477 bp DNA fragments at the 5 upstream region and 318 bp DNA fragments at the 3 downstream region) to be a molarity (M) of 2:1:1 according to the provided manual using Infusion Cloning Kit of TakaRa, a plasmid for mutant panB introduction was obtained. In order to secure Corynebacterium glutamicum-derived PLM1 promoter, PCR was performed using Corynebacterium glutamicum (ATCC13032) genome DNA as a template using primers of SEQ ID NOs: 108 and 109 as same as described above, to obtain promoter DNA fragments.

[0111] The information of the obtained mutant plasmids was summarized in Table 8 and Table 9 below:

TABLE-US-00008 TABLE 8 Mutant plasmid produced Mutation Amino acid to induce amino acid position substitution substitution Amino acid G116S pECCG117-panB(G116S) residue G116C pECCG117-panB(G116C) corresponding G116L pECCG117-panB(G116L) to the G116I pECCG117-panB(G116I) 116th G116T pECCG117-panB(G116T) residue of G116V pECCG117-panB(G116V) Wild type G116M pECCG117-panB(G116M) panB (SEQ G116D pECCG117-panB(G116D) ID NO: 37) G116E pECCG117-panB(G116E) G116N pECCG117-panB(G116N) G116Q pECCG117-panB(G116Q) G116A pECCG117-panB(G116A)

TABLE-US-00009 TABLE 9 Mutant plasmid produced Mutation Amino acid to induce amino acid position substitution substitution Amino acid A159R pECCG117-panB(A159R) residue A159S pECCG117-panB(A159S) corresponding A159Y pECCG117-panB(A159Y) to the A159C pECCG117-panB(A159C) 116th A159P pECCG117-panB(A159P) residue of A159H pECCG117-panB(A159H) Wild type A159L pECCG117-panB(A159L) panB (SEQ A159I pECCG117-panB(A159I) ID NO: 37) A159T pECCG117-panB(A159T) A159K pECCG117-panB(A159K) A159V pECCG117-panB(A159V) A159M pECCG117-panB(A159M) A159D pECCG117-panB(A159D) A159E pECCG117-panB(A159E) A159N pECCG117-panB(A159N) A159Q pECCG117-panB(A159Q) WT pECCG117-panB(WT)

Example 7. Evaluation of Pantothenic Acid Production Ability of 3-Methyl-2Ketobutanoate Hydroxymethyltransferase

[0112] The mutant plasmid produced in Example 6 (Table 8 and Table 9) and pECCG117-panB (WT-EC) (Table 1) were introduced into the ATCC13032 panB strain produced in Example 4 by an electric pulse method, and then they were smeared in a selective medium containing kanamycin 25 mg/l to obtain total 19 kinds of transformed mutant strains in which each random mutagenesis (saturated mutagenesis) was introduced. Then, by progressing flask evaluation by the same method as Example 3, the production ability of the obtained pantothenic acid was measured. The obtained result was shown in Table 10 and Table 11:

TABLE-US-00010 TABLE 10 Pantothenic acid (g/L) Arrangement Arrangement Arrangement Strain 1 2 3 Average ATCC 13032 panB (Control group) 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)

[0113] As could be seen in Table 10 above, the ATCC13032 panB strain did not produce pantothenic acid, whereas all the E. coli PanB (wild type) or mutant strains in which its mutant was introduced showed the pantothenic acid production ability. In addition, the mutant strains in which the G116S, G116C, G116L, G116I, G116T, G116V, G116D, G116E, G116N, G116A, G116M, or G116Q mutation was introduced produced pantothenic acid at a much higher level compared to ATCC 13032 panB pECCG117-panB (WT) which was the mutant strain comprising the E. coli PanB (wild type). As a result, both the wild type of E. coli PanB and mutant had the effect of increasing pantothenic acid production, and in particular, it could be confirmed that the 116th amino acid residue of PanB (SEQ ID NO: 37) was the important position for pantothenic acid production, and when the amino acid at this position was substituted with various amino acids different from the original one, the production ability of pantothenic acid was further increased.

[0114] The ATCC 13032 panB pECCG117-panB (G116A) strain (named Corynebacterium glutamicum CV03-5001) confirmed as having the most excellent production ability of pantothenic acid in the present example was deposited to Korean Culture Center of Microorganisms located in Hongje-dong, Seodaemun-gu, Seoul, Korea on Jun. 8, 2020 and was given an accession number of KCCM12744P.

TABLE-US-00011 TABLE 11 Pantothenic acid production ability of saturated mutagenesis-introduced strain Pantothenic acid (g/L) Arrangement Arrangement Arrangement Strain 1 2 3 Average ATCC 13032 0.0 0.0 0.0 0 ATCC 13032 pECCG117- 0.7 0.8 0.8 0.77 panB(A159R) ATCC 13032 pECCG117- 2.5 2.9 2.6 2.67 panB(A159S) ATCC 13032 pECCG117- 0.8 0.7 0.8 0.77 panB(A159Y) ATCC 13032 pECCG117- 2.2 2.3 2.2 2.23 panB(A159C) ATCC 13032 pECCG117- 1.2 1.3 1.3 1.27 panB(A159P) ATCC 13032 pECCG117- 0.7 0.8 0.7 0.73 panB(A159H) ATCC 13032 pECCG117- 2.8 3.1 2.9 2.93 panB(A159L) ATCC 13032 pECCG117- 2.5 2.7 2.6 2.6 panB(A159I) ATCC 13032 pECCG117- 2.1 2.1 2.3 2.17 panB(A159T) ATCC 13032 pECCG117- 0.9 0.6 0.7 0.73 panB(A159K) ATCC 13032 pECCG117- 2.6 2.7 2.4 2.57 panB(A159V) ATCC 13032 pECCG117- 1.9 1.9 2.2 2 panB(A159M) ATCC 13032 pECCG117- 1.3 1.1 1.2 1.2 panB(A159D) ATCC 13032 pECCG117- 0.8 0.8 0.7 0.77 panB(A159E) ATCC 13032 pECCG117- 1.2 1.5 1.6 1.43 panB(A159N) ATCC 13032 pECCG117- 1.2 1.1 1.2 1.17 panB(A159Q) ATCC 13032 pECCG117-panB(WT) 0.7 0.7 0.7 0.7

[0115] In addition, as could be confirmed in Table 11 above, the wild type Corynebacterium glutamicum ATCC 13032 strain did not produce pantothenic acid, whereas all the 19 kinds of mutant strains produced above showed the pantothenic acid production ability, and among them, the mutant strain in which the amino acid corresponding to the 159th residue of the wild type panB (SEQ ID NO: 37) was mutated into 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 high level, compared to the ATCC 13032 pECCG117-panB (WT) strain comprising the wild type panB. As a result, it could be confirmed that the amino acid residue corresponding to the 159th residue of panB (SEQ ID NO: 37) was the important position for pantothenic acid production, and it could be confirmed that when the amino acid at this position was substituted with various amino acids different from the original one, the production ability of pantothenic acid was further increased.

[0116] The ATCC 13032 panB pECCG117-panB (A159L) strain confirmed as having the most excellent production ability of pantothenic acid in the present example was named was CV03-5002 and was deposited to Korean Culture Center of Microorganisms located in Hongje-dong, Seodaemun-gu, Seoul, Korea on Apr. 13, 2021 and was given an accession number of KCCM12973P.

Example 8. Production and Evaluation of Mutant 3-Methyl-2Ketobutanoate Hydroxymethyltransferase with Enhanced Pantothenic Acid Production Ability

[0117] It was confirmed whether there was an additional activity improvement by combining the 3-methyl-2ketobutanoate hydroxymethyltransferase G116A variant and the 3-methyl-2ketobutanoate hydroxymethyltransferase A159L variant of which effect was confirmed in Example 7.

[0118] To produce a vector comprising the G116A and A159L variants, a vector comprising both G116A and A159L was produced using primers of SEQ ID NOs: 74, 126 and SEQ ID NOs: 77, 127 using pECCG117-panB (A159L) produced in Example 2 as a template in the same way as described in Example 2. The produced mutant plasmid pECCG117-panB (G116A, A159L) was introduced into the wild type Corynebacterium glutamicum ATCC13032 strain by an electric pulse method, and then was smeared into a selective medium containing kanamycin 25 mg/L to obtain each transformed strain. Then, by progressing flask evaluation by the same method as Example 2, the production ability of the obtained pantothenic acid was measured. The obtained result was shown in Table 12 below.

TABLE-US-00012 TABLE 12 Pantothenic acid production ability of mutant strain comprising G116A and A159L Pantothenic acid (g/L) Arrangement Arrangement Arrangement Strain 1 2 3 Average ATCC 13032 0.0 0.0 0.0 0 ATCC 13032 pECCG117-panB(WT) 0.7 0.7 0.6 0.67 ATCC 13032 pECCG117- 2.9 2.8 3.1 2.93 panB(A159L) ATCC 13032 pECCG117- 2.2 2.4 2.1 2.23 panB(G116A) ATCC 13032 pECCG117- 3.9 3.7 3.7 3.77 panB(G116A, A159L)

[0119] As could be confirmed in Table 12 above, the ATCC 13032 strain did not produce pantothenic acid, whereas the mutant strain comprising all the amino acid mutation corresponding to the 116th residue of panB and the amino acid mutation corresponding to the 159th residue produced pantothenic acid at a higher level compared to the ATCC 13032 pECCG117-panB (WT) strain comprising the wild type panB, and produced pantothenic acid at a higher level even than the mutant strains comprising an A159L or G116A mutation. As a result, it could be confirmed that the amino acids corresponding to the 116th and 159th residues of panB were important positions for pantothenic acid production, and it could be confirmed that when the amino acids at these positions were substituted with amino acids different from the original one, the production ability of pantothenic acid was further increased.

[0120] From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the examples described above are illustrative in all respects and not limitative. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described below and equivalent concepts rather than the detailed description above, in the scope of the present application.

[Accession Number]

[0121] Depository authority name: Korean Culture Center of Microorganisms [0122] Accession number: KCCM12744P [0123] Accession date: 20200608 [0124] Depository authority name: Korean Culture Center of Microorganisms [0125] Accession number: KCCM12973P [0126] Accession date: 20210413