MICROORGANISMS OF THE GENUS CORYNEBACTERIUM HAVING L-ISOLEUCINE PRODUCING ABILITY AND METHODS FOR PRODUCING L-ISOLEUCINE USING THE SAME

Abstract

The present application relates to a microorganism of the genus Corynebacterium having L-isoleucine producing ability which comprises a protein having an activity of citramalate synthase, and a method for producing L-isoleucine using the same.

Claims

1. A method for producing L-isoleucine, comprising: culturing a microorganism of the genus Corynebacterium comprising a protein having an activity of citramalate synthase in a medium; and recovering L-isoleucine from the microorganism or medium.

2. The method of claim 1, wherein the citramalate synthase is derived from a microorganism of the genus Methanocaldococcus.

3. The method of claim 1, wherein the citramalate synthase has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15.

4. The method of claim 1, wherein the microorganism of the genus Corynebacterium further comprises enhanced activities of 3-isopropylmalate dehydrogenase and 3-isopropylmalate dehydratase.

5. The method of claim 1, wherein the microorganism of the genus Corynebacterium further comprises inactivated pyruvate dehydrogenase.

6. The method of claim 1, wherein acetate is further provided in culturing the microorganism of the genus Corynebacterium.

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

8. A microorganism of the genus Corynebacterium having L-isoleucine producing ability, wherein the microorganism comprises a protein having an activity of citramalate synthase.

9. The microorganism of claim 8, wherein the citramalate synthase has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15.

10. The microorganism of claim 8, wherein the microorganism further comprises enhanced activities of 3-isopropylmalate dehydrogenase and 3-isopropylmalate dehydratase.

11. The microorganism of claim 8, wherein the microorganism further comprises inactivated pyruvate dehydrogenase.

12. The microorganism of claim 8, wherein the microorganism is Corynebacterium glutamicum.

Description

DETAILED DESCRIPTION OF EMBODIMENT

Example 1: Construction of a Recombinant Vector Including cimA Gene Derived from Methanocaldococcus

[0089] <1-1> Preparation of a Fragment of cimA Gene Derived from Methanocaldococcus

[0090] To obtain a 1476 bp fragment comprising the open reading frame (ORF) of cimA gene (NC_000909.1) encoding citramalate synthase, genomic DNA of Methanocaldococcus jannaschii DSM 2661 was extracted using a Genomic-tip system (Qiagen).

[0091] Citramalate synthase derived from Methanocaldococcus jannaschii DSM 2661 has the sequence of 491 amino acids represented by SEQ ID NO: 1, and cimA gene encoding the same has the nucleotide sequence of SEQ ID NO: 2.

[0092] Polymerase chain reaction (hereinafter abbreviated as PCR) was performed using the extracted genomic DNA (gDNA) as a template. The PCR was performed using the primer pair of SEQ ID NOS: 35 and 36 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0093] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a band having a desired size was eluted for recovery, and the product was labeled as cimA(M) fragment.

TABLE-US-00001 primercimA-5-NdeI(SEQIDNO:35): 5-GCATCATATGATGGTAAGGATATTC-3 primercimA-3-XbaI(SEQIDNO:36): 5-CGATCTAGATTAATTCAACAACATGTT-3

[0094] <1-2> Construction of Recombinant Vector p117-Cj7-cimA(M)

[0095] PCR was performed using p117-cj7-gfp including promoter cj7 derived from a known microorganism of the genus Corynebacterium (Korean Patent No. 10-0620092) as a template. As used herein, the term p117 represents pECCG117, which is an E. coli-Corynebacterium shuttle vector (Biotechnology Letters 13(10): 721-726, 1991). The PCR was performed using the primer pair of SEQ ID NOS: 27 and 28 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 20 sec, and elongation at 72 C. for 30 sec.

[0096] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 323 bp band was eluted for recovery, and the product was labeled as cj7 fragment.

TABLE-US-00002 primerPcj7-5-KpnI(SEQIDNO:27): 5-GATGGTACCACCCCAGAAACATCCCAGC-3 primerPcj7-3NdeI(SEQIDNO:28): 5-CGATCATATGGAGTGTTTCCTTTCGTTGGG-3

[0097] Sewing PCR was performed using the prepared cj7 fragment and cimA(M) fragment prepared in Example <1-1> above as a template. The sewing PCR was performed using the primer pair of SEQ ID NOS: 27 and 36 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0098] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 1799 bp band was eluted for recovery, and the product was labeled as cj7-cimA(M) fragment.

[0099] The obtained cj7-cimA(M) fragment was treated with restriction enzymes KpnI and XbaI, and then ligated with a linear p117 fragment treated with the same restriction enzymes.

[0100] E. coli DH5a cells were heat-shock transformed with the constructed vector, then plated on a 25 g/mL kanamycin-containing LB solid medium, and cultured overnight at 37 C. One platinum loop of the cultured colony was inoculated into 3 mL of a 25 g/mL kanamycin-containing LB liquid medium and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit (Catalogue No. 27104, Qiagen, hereinafter the same).

[0101] The construction of the recombinant vector was confirmed by treatment with restriction enzymes KpnI and XbaI, and the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 90 sec. The recovered recombinant vector was labeled as p117-cj7-cimA(M).

TABLE-US-00003 primer117-F(SEQIDNO:29): 5-CCACAGCCGACAGGATGGTGA-3 primer117-R(SEQIDNO:30): 5-CTCAGGGTGTAGCGGTTCGGT-3

[0102] <1-3> Preparation of a Fragment of leuBCD Gene Involved in the Biosynthesis Pathway of 2-Ketobutyrate

[0103] To prepare a recombinant vector for enhancing a gene in the biosynthesis pathway of 2-ketobutyrate, a fragment of the leuBCD gene encoding 3-isopropylmalate dehydrogenase and 3-isopropylmalate dehydratase derived from a microorganism of the genus Corynebacterium was prepared as described below.

[0104] To obtain a 1359 bp fragment comprising the ORF of leuB gene encoding 3-isopropylmalate dehydrogenase (EC1.1.1.85), genomic DNA of Corynebacterium glutamicum ATCC13032 was extracted using a Genomic-tip system (Qiagen).

[0105] 3-Isopropylmalate dehydrogenase derived from Corynebacterium glutamicum ATCC13032 has the sequence of 340 amino acids represented by of SEQ ID NO: 17, and leuB gene encoding the same has the nucleotide sequence of SEQ ID NO: 18.

[0106] PCR was performed using the extracted gDNA as a template. The PCR was performed using the primer pair of SEQ ID NOS: 31 and 32 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0107] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a band having a desired size was eluted for recovery, and the product was labeled as leuB fragment.

TABLE-US-00004 primerleuB-5-cimA(SEQIDNO:31): 5-TTGTTGAATTAATCTAGAGGTGACACCCCAGTGG-3 primerleuB-3-leuC(SEQIDNO:32): 5-TCGCAGCTGCCACCGATATTTAGCTTTGCAGCGC-3

[0108] To obtain a 2078 bp fragment comprising the ORF of leuCD gene encoding 3-isopropylmalate dehydratase (EC4.2.1.33), PCR was performed using gDNA of Corynebacterium glutamicum ATCC13032 as a template.

[0109] LeuC gene encodes a large subunit of 3-isopropylmalate dehydratase, and the leuC gene derived from Corynebacterium glutamicum ATCC13032 has the nucleotide sequence of SEQ ID NO: 20 encoding a polypeptide having the amino acid sequence of SEQ ID NO: 19. Further, leuD gene encodes a small subunit of 3-isopropylmalate dehydratase, and the leuD gene derived from Corynebacterium glutamicum ATCC13032 has the nucleotide sequence of SEQ ID NO: 22 encoding a polypeptide having the amino acid sequence of SEQ ID NO: 21.

[0110] The PCR was performed using the primer pair of SEQ ID NOS: 33 and 34 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 80 sec.

[0111] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a band having a desired size was eluted for recovery, and the product was labeled as leuCD fragment.

TABLE-US-00005 primerleuC-5-leuB(SEQIDNO:33): 5-GCGCTGCAAAGCTAAATATCGGTGGCAGCTGCGA-3 primerleuD-3-XbaI(SEQIDNO:34): 5-TGGCGGCCGCTCTAGAGCTTTCGCTATCAGACTG-3

[0112] Sewing PCR was performed using the leuB fragment and leuCD fragment recovered above as a template. The sewing PCR was performed using the primer pair of SEQ ID NOS: 31 and 34 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 120 sec.

[0113] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 3437 bp band was eluted for recovery, and the product was labeled as leuBCD fragment.

[0114] <1-4> Construction of Recombinant Vector p117-Cj7-cimA(M)-leuBCD

[0115] To prepare a recombinant vector for enhancing a gene in the biosynthesis pathway of 2-ketobutyrate, p117-cj7-cimA(M), which is the recombinant vector constructed in Example <1-2> above, was treated with restriction enzyme XbaI, the leuBCD fragment recovered in Example <1-3> above was treated with restriction enzyme XbaI, and then both fragments were ligated.

[0116] E. coli DH5 cells were heat-shock transformed with the constructed vector, then plated on a 25 g/mL kanamycin-containing LB solid medium, and cultured overnight at 37 C. One platinum loop of the cultured colony was inoculated into 3 mL of a 25 g/mL kanamycin-containing LB liquid medium and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit.

[0117] The construction of the recombinant vector was confirmed by treatment with restriction enzyme XbaI, and the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 180 sec. The recovered recombinant vector was labeled as p117-cj7-cimA(M)-leuBCD.

[0118] <1-5> Preparation of a Fragment of a cimA(M)Mutant Using Error-Prone PCR

[0119] To secure a DNA pool having a random mutation introduced to cimA gene, PCR was performed using the cimA(M) fragment recovered in Example <1-1> above as a template and the diversify PCR random mutagenesis kit (Catalogue No. 630703, Clonetech). The PCR was performed on condition 4 of the mutagenesis reaction in Table III in the user manual of the product using the primer pair of SEQ ID NOS: 35 and 36 under the following conditions: 25 cycles, each consisting of denaturation at 95 C. for 30 sec and elongation at 68 C. for 30 sec.

[0120] As a result of the PCR, a mutated art cimA DNA pool having a modification of nucleotide substitution randomly introduced was prepared. The PCR product (hereinafter, referred to as cimA(M)m fragment) was electrophoresed on 1.0% agarose gel then eluted for recovery.

TABLE-US-00006 primercimA-5-NdeI(SEQIDNO:35): 5-GCATCATATGATGGTAAGGATATTC-3 primercimA-3-XbaI(SEQIDNO:36): 5-CGATCTAGATTAATTCAACAACATGTT-3

[0121] <1-6> Construction of Mutant Library of p117-Cj7-cimA(M)m

[0122] Recombinant vector, p117-cj7-cimA(M)m, constructed in Example <1-2> above, was treated with restriction enzymes NdeI and XbaI, and was ligated together with the cimA(M)m fragment recovered in Example <1-5> above and treated with the same restriction enzymes.

[0123] E. coli DH5a cells were heat-shock transformed with the constructed vector, then plated on a 25 g/mL kanamycin-containing LB solid medium, and cultured overnight at 37 C. The cultured colonies were gathered, and then plasmid DNA was recovered using a plasmid miniprep kit thereby construct the mutant library of p117-cj7-cimA(M)m.

Example 2: Confirmation of L-Isoleucine Producing Ability by Introduction of cimA Gene

[0124] To confirm whether Corynebacterium glutamicum can has L-isoleucine producing ability when cimA gene derived from Methanocaldococcus is introduced thereto, a strain having ilvA gene removed encoding threonine dehydratase was constructed, and recovery of the L-isoleucine producing ability of the strain was examined by introducing the cimA gene thereto.

[0125] Threonine dehydratase is an enzyme converting L-threonine, which is a precursor of L-isoleucine biosynthesis, to 2-ketobutyrate. Threonine dehydratase derived from Corynebacterium glutamicum ATCC13032 has the amino acid sequence of SEQ ID NO: 23, and ilvA gene encoding the same has the nucleotide sequence of SEQ ID NO: 24. Further, any protein having an activity of threonine dehydratase is included in the scope of the present disclosure without limitation.

[0126] <2-1> Construction of ilvA Gene-Deleted Vector

[0127] To obtain an ilvA-gene fragment, PCR was performed using genomic DNA extracted from Corynebacterium glutamicum ATCC13032 using a Genomic-tip system (Qiagen) as a template. The PCR was performed using each primer pair of SEQ ID NOS: 37 and 38, and SEQ ID NOS: 39 and 40 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 45 sec.

[0128] The amplified PCR products were electrophoresed on 1.0% agarose gel, and then 547 bp and 467 bp bands for each PCR were eluted for recovery.

[0129] Sewing PCR was performed using the two fragments of the ilvA gene recovered above as a template. The sewing PCR was performed using the primer pair of SEQ ID NOS: 37 and 40 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0130] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 1014 bp band was eluted for recovery, and the recovered ilvA gene-deleted fragment was labeled as DilvA.

[0131] The DilvA fragment recovered above was treated with restriction enzyme XbaI, pDZ vector (Korean Patent No. 10-0924065) was treated with same restriction enzyme to recover a linear pDZ fragment, and then the DilvA and linear pDZ fragments were ligated to construct a recombinant vector having ilvA gene deleted.

[0132] E. coli DH5 cells were heat-shock transformed with the constructed recombinant vector, then plated on a 25 g/mL kanamycin-containing LB solid medium, and cultured overnight at 37 C. One platinum loop of the cultured colony was inoculated into 3 mL of a 25 g/mL kanamycin-containing LB liquid medium and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit.

[0133] The construction of the recombinant vector was confirmed by treatment with restriction enzyme XbaI, and the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 41 and 42 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 90 sec. The recovered recombinant vector was labeled as pDZ-ilvA(Del).

TABLE-US-00007 primerilvA-5-XbaI(SEQIDNO:37): 5-GCATCTAGAGAACACGGACAATGCCAC-3 primerilvA-Del-R(SEQIDNO:38): 5-CAAACAGCGTGATGTCCTGAGTGAGCTGCGCT-3 primerilvA-Del-F(SEQIDNO:39): 5-AGCGCAGCTCACTCAGGACATCACGCTGTTTG-3 primerilvA-3-XbaI(SEQIDNO:40): 5-GCATCTAGAACCTGCGGCACACCTTGGGC-3 primerTopo-F(SEQIDNO:41): 5-GCAGTGAGCGCAACGCAAT-3 primerTopo-R(SEQIDNO:42): 5-CGTTGTAAAACGACGGCCA-3

[0134] <2-2> Construction of ilvA Gene-Deleted Strain

[0135] The recombinant vector pDZ-ilvA(Del) constructed in Example <2-1> above was introduced into Corynebacterium glutamicum ATCC13032 as a parent strain by electroporation, and was plated on a solid medium containing 25 g/mL of kanamycin to select a single colony. From the colony, each strain having the pDZ-ilvA(Del) vector introduced into a chromosome was selected by the second passage, and then PCR was performed using gDNA recovered from each of the selected strains as a template. The PCR was performed using the primer pair of SEQ ID NOS: 43 and 44 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 90 sec.

[0136] As a result of the PCR, an ilvA gene-deleted strain was recovered, and was labeled as an ATCC13032ilvA strain.

TABLE-US-00008 primerCFilvA(SEQIDNO:43): 5-GATCTGTGATGAGGTGATG-3 primerCRilvA(SEQIDNO:44): 5-CGTCCTTCCATGACTCTCA-3

[0137] <2-3> Preparation of a Gene Fragment Involved in the Biosynthesis Pathway of L-Isoleucine

[0138] To prepare a recombinant vector for enhancing a gene in the biosynthesis pathway of L-isoleucine, fragments of lysC, asd, hom, thrB, thrC, and ilvA genes derived from a microorganism of the genus Corynebacterium were prepared as described below.

[0139] First, to obtain a DNA fragment comprising the ORF of lysC-asd gene encoding aspartate kinase and aspartate--semialdehyde dehydrogenase, PCR was performed using genomic DNA extracted from Corynebacterium glutamicum ATCC13032 as a template as described in Example <2-1>. The PCR was performed using the primer pair of SEQ ID NOS: 45 and 46 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 90 sec.

[0140] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 2666 bp band was eluted for recovery, and the product was labeled as lysC-asd fragment.

TABLE-US-00009 primerlysC-5-KpnI(SEQIDNO:45): 5-TGAGGTACCCATGCGATTGTTAATGC-3 primerasd-3-SfoI(SEQIDNO:46): 5-CTAGGCGCCAGTGTAGCACTCAAGCGGA-3

[0141] To obtain a DNA fragment comprising the ORF of hom-thrB gene encoding homoserine dehydrogenase and homoserine kinase, PCR was performed using genomic DNA extracted from Corynebacterium glutamicum ATCC13032 as a template as described in Example <2-1>. The PCR was performed using the primer pair of SEQ ID NOS: 47 and 48 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 90 sec.

[0142] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 2633 bp band was eluted for recovery, and the product was labeled as hom-thrB fragment.

TABLE-US-00010 primerhom-5-BamHI(SEQIDNO:47): 5-CTAGGATCCCTCACCATTCTCAATGGT-3 primerthrB-3-BamHI(SEQIDNO:48): 5-CTAGGATCCGCCTTCCTTGTTGGGC-3

[0143] To obtain a DNA fragment comprising the ORF of thrC gene encoding threonine synthase, PCR was performed using genomic DNA extracted from Corynebacterium glutamicum ATCC13032 as a template as described in Example <2-1>. The PCR was performed using the primer pair of SEQ ID NOS: 49 and 50 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0144] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 1703 bp band was eluted for recovery, and the product was labeled as thrC fragment.

TABLE-US-00011 primerthrC-5-SpeI(SEQIDNO:49): 5-TGCACTAGTGAGAACATACAGGTTCCA-3 primerthrC-3-ilvA(SEQIDNO:50): 5-GGCATTGTCCGTGTTCTTACTTCACGGAAGTG-3

[0145] To obtain a DNA fragment comprising the ORF of ilvA gene encoding threonine dehydratase, PCR was performed using genomic DNA extracted from Corynebacterium glutamicum ATCC13032 as a template as described in Example <2-1>. The PCR was performed using the primer pair of SEQ ID NOS: 51 and 52 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 60 sec.

[0146] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 1862 bp band was eluted for recovery, and the product was labeled as ilvA fragment.

TABLE-US-00012 primerilvA-5-thrC(SEQIDNO:51): 5-CACTTCCGTGAAGTAAGAACACGGACAATGCC-3 primerilvA-3-SpeI(SEQIDNO:52): 5-TGCACTAGTACCTGCGGCACACCTTGGGC-3

[0147] Sewing PCR was performed using the thrC fragment and ilvA fragment recovered above as a template. The sewing PCR was performed using the primer pair of SEQ ID NOS: 49 and 52 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 120 sec.

[0148] The amplified PCR product was electrophoresed on 1.0% agarose gel, then a 3565 bp band was eluted for recovery, and the product was labeled as thrC-ilvA fragment.

[0149] <2-4> Construction of a Vector for Enhancing a Gene in the Biosynthesis Pathway of L-Isoleucine

[0150] To prepare a recombinant vector for enhancing a gene in the biosynthesis pathway of L-isoleucine, the lysC-asd fragment obtained in Example <2-3> above was treated with restriction enzymes KpnI and SfoI, and then ligated with a linear p117 fragment treated with restriction enzymes KpnI and EcoRV.

[0151] A plasmid DNA was recovered in the same manner of Example <1-2> above. To confirm the construction of the recombinant vector, the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 100 sec. The recovered recombinant vector was labeled as p117-lysC-asd.

[0152] The hom-thrB fragment obtained in Example <2-3> above was treated with restriction enzyme BamHI, and then ligated with a linear p117-lysC-asd fragment treated with the same restriction enzyme.

[0153] A plasmid DNA was recovered in the same manner of Example <1-2> above. To confirm the construction of the recombinant vector, the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 180 sec. The recovered recombinant vector was labeled as p117-lysC-asd-hom-thrB.

[0154] The thrC-ilvA fragment obtained in Example <2-3> above was treated with restriction enzyme SpeI, and then ligated with a linear p117-lysC-asd-hom-thrB fragment treated with the same restriction enzyme.

[0155] A plasmid DNA was recovered in the same manner of Example <1-2> above. To confirm the construction of the recombinant vector, the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 300 sec.

[0156] The recovered recombinant vector was labeled as p117-IBGC (Isoleucine Biosynthesis Genes Cluster), which is a recombinant vector for enhancing a gene in the biosynthesis pathway of L-isoleucine including fragments of lysC, asd, hom, thrB, thrC, and ilvA genes, six genes involved in the biosynthesis of L-isoleucine.

[0157] <2-5> Comparison of L-Isoleucine Productivity

[0158] Recombinant vectors p117-cj7-cimA(M), p117-cj7-cimA(M)-leuBCD, and p117-IBGC constructed in Examples <1-2>, <1-4>, and <2-4> above were introduced into Corynebacterium glutamicum ATCC13032 by electroporation, and were plated on a solid medium containing 25 g/mL of kanamycin to select a single colony. From the colony, each of the selected strains was labeled as Corynebacterium glutamicum ATCC13032/p117-cj7-cimA(M), ATCC13032/p117-cj7-cimA(M)-leuBCD, and ATCC13032/p117-IBGC.

[0159] Further, p117-cj7-cimA(M) and p117-cj7-cimA(M)-leuBCD were introduced into a Corynebacterium glutamicum ATCC13032ilvA strain prepared in Example <2-2> above by electroporation, and were plated on a solid medium containing 25 g/mL of kanamycin to select a single colony. From the colony, each of the selected strains was labeled as Corynebacterium glutamicum ATCC13032ilvA/p117-cj7-cimA(M) and ATCC13032ilvA/p117-cj7-cimA(M)-leuBCD.

[0160] Titer evaluation on L-isoleucine productivity was performed using Corynebacterium glutamicum ATCC13032, ATCC13032/p117-IBGC, ATCC13032/p117-cj7-cimA(M), ATCC13032/p117-cj7-cimA(M)-leuBCD, ATCC13032ilvA, ATCC13032ilvA/p117-cj7-cimA(M), and ATCC13032ilvA/p117-cj7-cimA(M)-leuBCD.

[0161] Specifically, each of the strains was inoculated into a 250 mL flask containing 25 mL of a glucose-containing titer medium having the composition shown in Table 1 below, and was then cultured in an incubator at 32 C. and 200 rpm for 30 hours. The concentrations of L-isoleucine produced are shown in Table 2 below.

TABLE-US-00013 TABLE 1 Composition Concentration (per liter) Glucose 60 g KH.sub.2PO.sub.4 1.1 g (NH.sub.4).sub.2SO.sub.4 20 g MgSO.sub.47H.sub.2O 1.2 g FeSO.sub.47H.sub.2O 90 mg MnSO.sub.44H.sub.2O 90 mg Thiamine-HCl 4.5 mg d-Biotin 0.9 mg HSM 20 g Calcium carbonate 30 g pH 7.0

TABLE-US-00014 TABLE 2 Strain L-isoleucine (g/L) ATCC13032 0.02 ATCC13032/p117-IBGC 0.70 ATCC13032/p117-cj7-cimA(M) 0.83 ATCC13032/p117-cj7-cimA(M)-leuBCD 1.25 ATCC13032ilvA 0.00 ATCC13032ilvA/p117-cj7-cimA(M) 0.08 ATCC13032ilvA/p117-cj7-cimA(M)-leuBCD 0.15

[0162] As disclosed in Table 2 above, a wild-type strain ATCC13032 having ilvA gene produced 0.02 g/L of L-isoleucine, and ATCC13032/p117-IBGC, a transformed strain having the genes in the biosynthesis pathway of L-isoleucine concurrently introduced, produced 0.70 g/L of L-isoleucine, showing that L-isoleucine productivity increased by 3400% compared with the wild-type strain.

[0163] Further, transformed strains ATCC13032/p117-cj7-cimA(M) and ATCC13032/p117-cj7-cimA(M)-leuBCD produced 0.83 g/L and 1.25 g/L of L-isoleucine, respectively, showing that L-isoleucine productivity increased by 4050% and 6150%, respectively, compared with the wild-type strain.

[0164] From the comparison, the L-isoleucine productivity of ATCC13032/p117-cj7-cimA(M), a strain transformed to introduce a cimA gene into the existing biosynthesis pathway of L-isoleucine, was confirmed as being superior to that of ATCC13032/p117-IBGC, a strain transformed to enhance the biosynthesis pathway of L-isoleucine itself. Further, the L-isoleucine productivity of ATCC13032/p117-cj7-cimA(M)-leuBCD, a transformed strain further enhanced with leuBCD gene, was confirmed as being further capable.

[0165] Meanwhile, the ilvA gene-deleted ATCC13032ilvA strain lost the L-isoleucine productivity, whereas the ATCC13032ilvA/p117-cj7-cimA(M) strain having cimA gene introduced produced 0.08 g/L of L-isoleucine, and the ATCC13032ilvA/p117-cj7-cimA(M)-leuBCD strain having cimA gene introduced and a leuBCD gene enhanced produced 0.15 g/L thereof.

[0166] The above results show that producing L-isoleucine is possible using a novel biosynthesis pathway of L-isoleucine by the introduction of cimA gene, not the existing biosynthesis pathway of L-isoleucine using L-threonine as a precursor.

Example 3: Preparation of a Transformed Recombinant Strain and Comparison of L-Isoleucine Productivity

[0167] <3-1> Preparation of a Recombinant Strain Using a Wild-Type Microorganism of the Genus Corynebacterium

[0168] To provide acetyl-CoA used as a precursor of cimA gene, acetate-auxotrophic strain of wild-type Corynebacterium glutamicum ATCC13032 in which aceE gene encoding pyruvate dehydrogenase is deleted was prepared, and was labeled as ATCC13032aceE (Schreiner et al., J Bacteriol. 187: 6005-18, 2005).

[0169] Pyruvate dehydrogenase derived from Corynebacterium glutamicum ATCC13032 has the amino acid sequence of SEQ ID NO: 25, and aceE gene encoding the same has the nucleotide sequence of SEQ ID NO: 26.

[0170] Recombinant vector p117-cj7-cimA(M) and mutant library of p117-cj7-cimA(M)m prepared in Example 1 above were introduced into the ATCC13032aceE strain prepared above by electroporation. The constructed strains were labeled as ATCC13032aceE/p117-cj7-cimA(M) and ATCC13032aceE/p117-cj7-cimA(M)m mutant library, respectively.

[0171] The ATCC13032aceE, ATCC13032aceE/p117-cj7-cimA(M), and ATCC13032aceE/p117-cj7-cimA(M)m mutant library were plated on a solid medium containing 25 g/mL of kanamycin to select a single colony, and titer evaluation on L-isoleucine productivity was performed using the selected strains.

[0172] Specifically, the strains were inoculated into a 250 mL flask containing 25 mL of a titer medium containing acetate in the form of ammonium acetate having the composition shown in Table 3 below, and then were cultured in an incubator at 32 C. and 200 rpm for 30 hours. As a result, seven types of colonies having increased L-isoleucine concentration were selected, and the list and L-isoleucine concentrations thereof are shown in Table 4 below.

TABLE-US-00015 TABLE 3 Composition Concentration (per liter) Glucose 60 g C.sub.2H.sub.7NO.sub.2 5 g KH.sub.2PO.sub.4 1.1 g (NH.sub.4).sub.2SO.sub.4 20 g MgSO.sub.47H.sub.2O 1.2 g FeSO.sub.47H.sub.2O 90 mg MnSO.sub.44H.sub.2O 90 mg Thiamine-HCl 4.5 mg d-Biotin 0.9 mg HSM 20 g Calcium carbonate 30 g pH 7.0

TABLE-US-00016 TABLE 4 Strain L-isoleucine (g/L) ATCC13032aceE 0.05 ATCC13032aceE/p117-cj7-cimA(M) 0.84 ATCC13032aceE/p117-cj7-cimA(M)m1 1.02 ATCC13032aceE/p117-cj7-cimA(M)m2 1.35 ATCC13032aceE/p117-cj7-cimA(M)m3 2.23 ATCC13032aceE/p117-cj7-cimA(M)m4 1.24 ATCC13032aceE/p117-cj7-cimA(M)m5 0.96 ATCC13032aceE/p117-cj7-cimA(M)m6 1.73 ATCC13032aceE/p117-cj7-cimA(M)m7 0.97

[0173] As a result, as disclosed in Table 4 above, the ATCC13032aceE strain as a parent strain produced 0.05 g/L of L-isoleucine, and transformed strains produced 0.84 g/L to 2.23 g/L of L-isoleucine, showing that the L-isoleucine productivity increased by 1580% to 4360%, respectively, compared with the parent strain. In particular, transformed strain ATCC13032aceE/p117-cj7-cimA(M)m3 recorded the highest L-isoleucine productivity among the strains compared with the parent strain ATCC13032aceE.

[0174] To identify the mutation site of the mutants of cimA(M) gene selected by the increased productivity of L-isoleucine shown in Table 4, and the substituted amino acids of each mutation, sequencing was performed.

[0175] As a result, as the CimA(M)m mutant library,

[0176] CimA(M)m1 has the amino acid sequence of SEQ ID NO: 3, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 4;

[0177] CimA(M)m2 has the amino acid sequence of SEQ ID NO: 5, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 6;

[0178] CimA(M)m3 has the amino acid sequence of SEQ ID NO: 7, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 8;

[0179] CimA(M)m4 has the amino acid sequence of SEQ ID NO: 9, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 10;

[0180] CimA(M)m5 has the amino acid sequence of SEQ ID NO: 11, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 12;

[0181] CimA(M)m6 has the amino acid sequence of SEQ ID NO: 13, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 14; and

[0182] CimA(M)m7 has the amino acid sequence of SEQ ID NO: 15, and may be encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO: 16.

[0183] <3-2> Preparation of a Recombinant Strain Using p117-Cj7-cimA(M)m3 Having the Highest Activity of the Selected p117-cimA(M)m Mutant Library

[0184] The p117-cj7-cimA(M)m3 vector identified as having the highest L-isoleucine productivity in Example <3-1> above was treated with XbaI, and was ligated with a DNA fragment recovered by treating a leuBCD fragment recovered in Example <1-3> above with the same restriction enzyme.

[0185] E. coli DH5a cells were heat-shock transformed with the constructed vector, then plated on a 25 g/mL kanamycin-containing LB solid medium, and cultured overnight at 37 C. One platinum loop of the cultured colony was inoculated into 3 mL of a 25 g/mL kanamycin-containing LB liquid medium and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit.

[0186] The construction of the recombinant vector was confirmed by treatment with restriction enzyme XbaI, and the clone was identified by performing PCR using the primer pair of SEQ ID NOS: 29 and 30 under the following conditions: 30 cycles, each consisting of denaturation at 95 C. for 30 sec, annealing at 56 C. for 30 sec, and elongation at 72 C. for 180 sec. The recovered recombinant vector was labeled as p117-cj7-cimA(M)m3-leuBCD.

[0187] Each of 17-cj7-cimA(M) of Example <1-2>, p117-cj7-cimA(M)-leuBCD of Example <1-4>, p117-cj7-cimA(M)m3 of Example <3-1>, and p117-cj7-cimA(M)m3-leuBCD of Example <3-2> above was introduced into the ATCC13032aceE strain used in Example <3-1> by electroporation, and was plated on a solid medium containing 25 g/mL of kanamycin to select a single colony.

[0188] The selected strains were inoculated into a 250 mL flask containing 25 mL of a titer medium containing glucose having the composition shown in Table 3 above, and then were cultured in an incubator at 32 C. and 200 rpm for 30 hours to examine the L-isoleucine productivity. The results are shown in Table 5 below.

TABLE-US-00017 TABLE 5 Strain L-isoleucine (g/L) ATCC13032aceE 0.04 ATCC13032aceE/p117-cj7-cimA(M) 0.98 ATCC13032aceE/p117-cj7-cimA(M)-leuBCD 2.09 ATCC13032aceE/p117-cj7-cimA(M)m3 2.22 ATCC13032aceE/p117-cj7-cimA(M)m3-leuBCD 3.76

[0189] As disclosed in Table 5 above, while the ATCC13032aceE strain as a parent strain produced 0.04 g/L of L-isoleucine, transformed strain ATCC13032aceE/p117-cj7-cimA(M) produced 0.98 g/L of L-isoleucine and ATCC13032aceE/p117-cj7-cimA(M)-leuBCD produced 2.09 g/L of L-isoleucine, showing increases of 0.94 g/L and 2.05 g/L compared with the L-isoleucine productivity of the parent strain.

[0190] Further, transformed strains ATCC13032aceE/p117-cj7-cimA(M)m3 and ATCC13032aceE/p117-cj7-cimA(M)m3-leuBCD including the cimA(M)m3 mutant gene recovered in Example <3-1> produced 2.22 g/L and 3.76 g/L of L-isoleucine, respectively, which increased by 5450% and 9300% of the production compared with the parent strain. Accordingly, this result proves that the cimA(M)m3 mutant is more effective than a wild-type strain thereof in the increase of the L-isoleucine productivity.

[0191] Among the transformed strains recovered in the above Examples, ATCC13032aceE/p117-cj7-cimA(M)m3 was labeled as Corynebacterium glutamicum CA10-1002, deposited to the Korean Culture Center of Microorganisms (KCCM) under the Budapest Treaty on Feb. 27, 2015, and was provided with Accession No. KCCM11672P.

[0192] <3-3> Preparation of a Recombinant Strain Using L-Isoleucine-Producing Strain KCCM11248P

[0193] Each of p117-cj7-cimA(M), p117-cj7-cimA(M)-leuBCD, p117-cj7-cimA(M)m3, and p117-cj7-cimA(M)m3-leuBCD, the recombinant vectors used in Examples <3-1> and <3-2>, was introduced into Corynebacterium glutamicum KCCM11248P, an L-isoleucine-producing strain (Korean Patent No. 10-1335789), and was plated on a solid medium containing 25 g/mL of kanamycin to select a single colony.

[0194] Each of the selected strains was labeled as KCCM11248P/p117-cj7-cimA(M), KCCM11248P/p117-cj7-cimA(M)-leuBCD, KCCM11248P/p117-cj7-cimA(M)m3, and KCCM11248P/p117-cj7-cimA(M)m3-leuBCD.

[0195] The L-isoleucine productivity of the strains was examined using L-isoleucine titer media having the composition shown in Table 6 below and culturing the strains in flasks as described below.

TABLE-US-00018 TABLE 6 Composition Concentration (per liter) Glucose 45 g BM 10 g KH.sub.2PO.sub.4 1.1 g (NH.sub.4).sub.2SO.sub.4 12.5 g MgSO.sub.47H.sub.2O 1.2 g FeSO.sub.47H.sub.2O 180 mg MnSO.sub.44H.sub.2O 180 mg ZnSO.sub.45H.sub.2O 0.9 mg CuSO.sub.45H.sub.2O 0.9 mg Thiamine-HCl 9 mg d-Biotin 1.8 mg Ca-Pantothenate 9 mg NCA 60 mg HSM 20 g Calcium carbonate 30 g pH 7.0

[0196] Specifically, parent strain KCCM11248P and transformed strains KCCM11248P/p117-cj7-cimA(M), KCCM11248P/p117-cj7-cimA(M)-leuBCD, KCCM11248P/p117-cj7-cimA(M)m3, and KCCM11248P/p117-cj7-cimA(M)m3-leuBCD were inoculated into 250 mL flasks containing 25 mL of titer media containing glucose having the composition shown in Table 6, and then were cultured in an incubator at 32 C. and 200 rpm for 60 hours to examine the L-isoleucine productivity. The results are shown in Table 7 below.

TABLE-US-00019 TABLE 7 Strain L-isoleucine (g/L) KCCM11248P 3.33 KCCM11248P/p117-cj7-cimA(M) 4.04 KCCM11248P/p117-cj7-cimA(M)-leuBCD 4.82 KCCM11248P/p117-cj7-cimA(M)m3 4.71 KCCM11248P/p117-cj7-cimA(M)m3-leuBCD 11.21

[0197] As disclosed in Table 7 above, while the parent strain KCCM11248P produced 3.33 g/L of L-isoleucine, transformed strains KCCM11248P/p117-cj7-cimA(M) produced 4.04 g/L of L-isoleucine, and KCCM11248P/p117-cj7-cimA(M)-leuBCD produced 4.82 g/L of L-isoleucine, showing increases of 0.71 g/L and 1.49 g/L compared with the L-isoleucine productivity of the parent strain.

[0198] Meanwhile, the transformed strains KCCM11248P/p117-cj7-cimA(M)m3 and KCCM11248P/p117-cj7-cimA(M)m3-leuBCD produced 4.71 g/L and 11.21 g/L of L-isoleucine, respectively, which increased in production by 41.4% and 236.6% compared with the parent strain.

[0199] As shown in the result of Example <3-2> using Corynebacterium glutamicum ATCC13032, this result further proves that the cimA(M)m3 mutant is more effective than a wild-type strain thereof in the increase of the L-isoleucine productivity.

[0200] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.