Microorganism for producing L-amino acid having increased cytochrome C activity, and L-amino acid production method using same

Abstract

Provided are a microorganism for producing L-amino acid, having increased cytochrome C activity, and an L-amino acid production method using the microorganism.

Claims

1. An L-amino acid producing microorganism having enhanced activity of cytochrome C, wherein the cytochrome C is cytochrome C-551 derived from Bacillus pseudofirmus OF4, wherein the cytochrome C-551 comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27, and wherein the L-amino acid producing microorganism is the genus of Corynebacterium.

2. The L-amino acid producing microorganism of claim 1, having an increased sugar consumption rate, compared to a homogeneous microorganism in which the activity of cytochrome C is not enhanced.

3. The L-amino acid producing microorganism of claim 1, having an improved L-amino acid production potential, compared to a homogeneous microorganism in which the activity of cytochrome C is not enhanced.

4. The L-amino acid producing microorganism of claim 3, wherein the L-amino acid is L-lysine.

5. A method for producing an L-amino acid, the method comprising the steps of: culturing the L-amino acid producing microorganism of claim 1 in a medium; and recovering the L-amino acid from the cultured microorganism, the medium, or both of them.

6. The method of claim 5, wherein the L-amino acid is L-lysine.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram accounting for analysis results of nucleic acid sequences of the library vectors prepared in an embodiment.

MODE FOR INVENTION

(2) 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 obvious 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: Construction of Vector Library for Gene Delivery

(3) In order to investigate genes useful for enhancing the lysine production potential of Corynebacterium glutamicum, a library of genomic DNAs derived from extremophile bacteria, which adapt to and survive various extreme environments, was constructed. As extremophile bacteria, which can grow under the extreme conditions of high osmotic pressures, high temperatures, hypoxia, and various hydrogen ion concentrations, the four representative microorganisms, Bacillus atrophaeus (ATCC 49337), Bacillus licheniformis (KCTC 1030), Lactobacillus fermentum (KCTC 3112), and Bacillus pseudofirmus OF4 (ATCC BAA2126) were used.

(4) First, genomic DNAs were extracted from the four strains, using a QlAamp DNA Micro Kit (QIAGEN). The genomic DNAs thus procured were digested with the restriction enzyme Sau3A1 (NEB) at 37 C. for 10 min and then at 65 C. for 30 min to give incomplete gene fractions which were then run on 1% agarose gel by electrophoresis. Only the gel fraction in the band of 5 to 7 kb were excised. From the gel, the gene fragments for insertion were eluted using GeneAll Expin GEL SV kit (Seoul, KOREA).

(5) The gene fragments thus procured were incubated with the restriction enzyme BamHI-HF (NEB) at 37 C. for one hour and then with CIP (NEB) at 37 C. for 30 min before ligation to the pECCG117 vector (Korean Patent No. 0057684). The resulting recombinant vector was transformed into E. coli DH5a which was then spread on an LB plate containing kanamycin (25 mg/l). Genes from a single colony were amplified by PCR using the primers of SEQ ID NOS: 1 and 2 shown in Table 1, below. PCR was started with an initial 10 min denaturing at 95 C. and proceeded with 30 cycles of denaturing at 95 C. for 1 min, annealing at 55 C. for 1 min, and extension at 72 C. for 4 min, followed by final extension at 72 C. for 10 min.

(6) TABLE-US-00001 TABLE1 Description sequence(5>3) SEQIDNO: Fprimer TAATACGACTCACTATAGGG 1 Rprimer CAATTAACCCTCACTAAA 2

(7) Detection of the PCR products confirmed that extremophile bacterium-derived genomic DNA fragments having a size of 3 to 5 kb were successfully inserted into the pECCG117 vector at a rate of 99% or higher. As many as 10,000 colonies of the transformants were secured per strain. From the transformants, plasmids were extracted using a plasmid prep kit (QIAGEN). The library vectors were called p117-Lib.Bat (derived from Bacillus atrophaeus), p117-Lib.Bli (derived from Bacillus licheniformis), p117-Lib.Lfe (derived from Lactobacillus fermentum), and p117-Lib.Bps (derived from Bacillus pseudofirmus OF4).

Example 2: Generation and Screening of Strain Having Vector Library Introduced Thereinto

(8) The four library vectors (p117-Lib.Bat, p117-Lib.Bli, p117-Lib.Lfe, and p117-Lib.Bps) constructed in Example 1 were transformed into the lysine producing strain Corynebacterium glutamicum KCCM11016P (Korean Patent No. 10-0159812) by an electric pulse method (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999) and the bacteria was spread on a complex plate medium containing kanamycin (25 mg/l). Finally, about 5,000 colonies per library vector were procured and named LYS_Lib.Bat, LYS_Lib.Bli, LYS_Lib.Lfe, and LYS_Lib.Bps, respectively. The KCCM11016P strain which had been transformed with the pECCG117 vector carrying no gDNA-derived gene fragments was used as a control against the library strains and named LYS_117 control.

(9) The composition of the complex plate medium used was as follows:

(10) <Complex Plate Medium (pH 7.0)>

(11) Glucose 10 g, Peptone 10 g, Beef extract 5 g, Yeast extract 5 g, Brain Heart Infusion 18.5 g, NaCl 2.5 g, Urea 2 g, Sorbitol 91 g, Agar 20 g (per liter of distilled water)

(12) The four KCCM11016P-based library strains procured were each inoculated into 96-Deep Well Plate-Dome (Bioneer) containing 400 l of a screening medium, using the colony-picker (SINGER, PIXL) and incubated in a plate shaking incubator (TAITEC) at 32 C. for 15 hr while shaking at 12,000 rpm.

(13) The seed medium has the following composition:

(14) <Screening Medium (pH 7.0)>

(15) Glucose 45 g, Sugar beet-derived molasses 10 g, Soybean steep liquid 10 g, (NH.sub.4).sub.2SO.sub.424 g, MgSO.sub.4.Math.7H.sub.2O 0.6 g, KH.sub.2PO.sub.40.55 g, Urea 5.5 g, Biotin 0.9 mg, Thiamine HCl 4.5 mg, Calcium pantothenate 4.5 mg, Nicotinamide 30 mg, MnSO.sub.4.Math.5H.sub.2O 9 mg, ZnSO.sub.4.Math.5H.sub.2O 0.45 mg, CuSO.sub.4.Math.5H.sub.2O 0.45 mg, FeSO.sub.4.Math.5H.sub.2O 9 mg, and Kanamycin 25 mg (per liter of distilled water)

(16) While being cultured, the cells were monitored for growth with the aid of a microplate-reader (BioTek). Concentrations of glucose and produced lysine in the media were measured using a sugar analyzer and (YSI) and a HPLC instrument (Shimadzu), respectively.

(17) Through the experiment, final selection was made of three strains that exhibits excellent growth and high glucose consumption rates, compared to the control (Table 2). The three selected strains were found to anchor the LYS_Lib.Bps library vectors thereat. Although being similar to the control to LYS_117 control in terms of yield and OD value, these strains were found excellent in productivity because their glucose consumption rates per hour (g/hr) in sampling point sections were increased by 118% relative to 100% of LYS_117 control.

(18) TABLE-US-00002 TABLE 2 Relative Sugar 36-Hr OD 600 Consumption Lysine Strain 12 hr 36 hr Rate (%) Yield (%) LYS_117 control 17.1 62.7 100 15.8 LYS_Lib.Bps #257 17.4 63.5 117.4 15.9 LYS_Lib.Bps #881 16.8 64.1 111.1 15.6 LYS_Lib.Bps #4213 18.1 63.8 118.0 15.8

Example 3: Base Sequencing of gDNA Library

(19) To identify sequences of the genes introduced into the three colonies selected in Example 2, LYS_Lib.Bps #257, #881, and #4213, gDNA library gene fragments that the colonies contained were amplified by PCT using the primers of SEQ ID NOS: 1 and 2 shown in Table 1 of Example 1. PCR was performed in the same condition as in Example 1. The PCR fragments were isolated using the GeneAll Expin GEL SV kit (Seoul, KOREA) and analyzed for base sequences. Based on the analysis results, gene information was obtained by BLAST (NCBI reference sequence NC_013791.2).

(20) The analysis results are depicted in FIG. 1. As shown in FIG. 1, the base sequencing result informed that there are a 4794-bp gene fragment in the colony LYS_Lib.Bps #257, a 3985-bp gene fragment in the colony #881, and a 4483-bp fragment in the colony #4213. The three colonies were found to have BpOF4_13735 and BpOF4_13740 as intact gene ORFs in common with one another. Subsequently, additional experiments were performed for influences of the two genes.

Example 4: Construction of Vector and Strain Having Individual Gene Introduced Thereinto

(21) For use in investing influences of the two individual genes identified in Example 3, a genomic insertion vector was constructed. For use as a base vector for gene insertion, first, pDZ_2284 vector targeting Ncgl2284, which is one of transposases, was constructed.

(22) In detail, ATCC13032 gDNA was used as a DNA template for PCR. Primers were prepared with reference to the NCBI base sequence (NC_003450.3). In the presence the primers of SEQ ID NOS: 3 and 4, PCR was started with 10 min denaturing at 95 C. and proceeded with 30 cycles of denaturing at 95 C. for 1 min, annealing at 55 C. for 1 min, and extension at 72 C. for 4 min, followed by final extension at 72 C. for 10 min to afford a 5 DNA fragment about 900 bp long. Likely, PCR was conducted using primers of SEQ ID NOS: 5 and 6 in the same condition as in the foregoing to amplify a 3 DNA fragment. The two DNA amplicons were purified using GeneAll Expin GEL SV kit (Seoul, KOREA) and digested with the restriction enzyme Xbal (NEB). Using an infusion cloning kit, the digest was ligated to pDZ (Korean Patent No. 2009-0094433) that has been thermally treated at 65 C. for 20 min. The recombinant plasmid thus formed was transformed into E. coli DH5a which was then spread on an LB plate medium containing kanamycin (25 mg/l). The gene inserted into pDZ vector was subjected to base sequencing to finally prepare pDZ_2284 vector.

(23) An additional enrichment (expression) vector for individual genes was constructed by digesting the base vector pDZ_2284 with the restriction enzymes Ndel and CIP (NEB), thermally treating the digested vector at 65 C. for 20 min, purifying the thermally treated vector, and ligating a promoter and each gene DNA fragment to the vector with the aid of an Infusion Cloning Kit. As the promoter for the additional gene expression, the gapA gene promoter of SEQ ID NO: 13 was employed. In order to obtain the promoter, PCR was performed using primers of SEQ ID NOS: 7 and 8, with ATCC13032 gDNA (NC_003450.3) serving as a template. In the presence of pfu polymerase, PCR was started with 10 min denaturing at 95 C. and proceeded with 30 cycles of denaturing at 95 C. for 1 min, annealing at 55 C. for 1 min, and extension at 72 C. for 1 min, followed by final extension at 72 C. for 10 min. DNA fragments for the two genes BpOF4_13735 and BPOF4_13740 were amplified from Bacillus pseudofirmus OF4 gDNA in the same manner as for the promoter, with the exception of using the primers of SEQ ID NOS: 9 and 10 for BpOF4_13735 (SEQ ID NO: 14) and the primers of SEQ ID NO: 11 and 12 for BpOF4_13740 (SEQ ID NO: 15). The DNA fragments thus obtained were purified using GeneAll Expin GEL SV kit (Seoul, KOREA) and then ligated to pDZ_2284 to finally afford two different vectors: pDZ_2284::PgapA BpOF4_13735 and pDZ_2284::PgapA BpOF4_13740.

(24) The two vector constructs were each transformed into the lysine producing strain Corynebacterium glutamicum KCCM11016P (Korean Patent No. 10-0159812), using an electric pulse method. Secondary DNA-crossover enriched the individual genes in the strains. The two final strains thus constructed were named KCCM11016P_2284::PgapA BpOF4_13735 and KCCM11016P_2284::PgapA BpOF4_13740, respectively.

(25) The primers, promoters, nucleic acid sequences of BpOF4 genes, and amino acid sequences encoded by the genes used herein are summarized in Table 3, below:

(26) TABLE-US-00003 TABLE3 Description Sequence(5.fwdarw.3orN.fwdarw.C) SEQIDNO Fprimerfor GTACCCGGGGATCCTCTAGAATCGCAATGATAGCCCATTC 3 ATCC13032gDNA Rprimerfor TTGGTCAAACCTCCCCTcatatgCAGAAATCCACATCAAT 4 ATCC13032gDNA Fprimerfor ATTGATGTGGATTTCTGcatatgAGGGGAGGTTTGACCAA 5 ATCC13032gDNA Rprimerfor GCCTGCAGGTCGACTCTAGAATGCATCTCTGGATGATGTG 6 ATCC13032gDNA FprimerforgapA ATTGATGTGGATTTCTGcatAAGCCTAAAAACGACCGAGC 7 promoter RprimerforgapA GTTGTGTCTCCTCTAAAGATTGTAG 8 promoter Fprimerfor ATCTTTAGAGGAGACACAACATGGATGAAAAAAGAAAAGC 9 BpOF4_13735 Rprimerfor TTGGTCAAACCTCCCCTcatTTAACGCCCCAGCCAAAAAATTCC 10 BpOF4_13735 Fprimerfor ATCTTTAGAGGAGACACAACATGAAAGGAAGACCACTTTT 11 BpOF4_13740 Rprimerfor TTGGTCAAACCTCCCCTcatTTATTCTGAAATAGATAGTA 12 BpOF4_13740 gapApromoter AAGCCTAAAAACGACCGAGCCTATTGGGATTACCATTGAAGCCA 13 GTGTGAGTTGCATCACATTGGCTTCAAATCTGAGACTTTAATTT GTGGATTCACGGGGGTGTAATGTAGTTCATAATTAACCCCATTC GGGGGAGCAGATCGTAGTGCGAACGATTTCAGGTTCGTTCCCTG CAAAAACTATTTAGCGCAAGTGTTGGAAATGCCCCCGTTTGGGG TCAATGTCCATTTTTGAATGTGTCTGTATGATTTTGCATCTGCT GCGAAATCTTTGTTTCCCCGCTAAAGTTGAGGACAGGTTGACAC GGAGTTGACTCGACGAATTATCCAATGTGAGTAGGTTTGGTGCG TGAGTTGGAAAAATTCGCCATACTCGCCCTTGGGTTCTGTCAGC TCAAGAATTCTTGAGTGACCGATGCTCTGATTGACCTAACTGCT TGACACATTGCATTTCCTACAATCTTTAGAGGAGACACAAC BpOF4_13735 ATGGATGAAAAAAGAAAAGCGATTATTATAAATGAAATTAAGTA 14 nucleicacidsequence CTGGCGCGAATCAAAGCTGCTTCCCTCCCAGTATTGTGATTTCT TATTAACGCTTTATTCAGAAGGAGAGGACCTAGAGACAGCCGAC TCAGGAAAGCGCTTCCGAAACATTCGGACAATCTATTCGTTTAT TATTGTTCAGCTTTCATTTGTCTTTACTGCTCTTGTCATTTATT TTACTGATTTTTCAAATGGATTGCAAATGCTTATTGGTTTGACT TTTTCGATTATTGTGTTAATTATAGCAAAACGGACTAGGGCAGA TGCCTTTTTTCTTAAACAATTTTACTATTTTATAGGGGCTCTGA TCCTCTTTTTACTAACGATTGAATGGGTTGTTCACTACAAAAGT ACTAATAACCTTTTATTATCAGCAACAATCATTTTACATTGCGT TTTTTGGCTCTTTGCAGGGCTGAAATGGAAAATGCGATTTTTTA CGATATCTGCTATACTAGGACTAGTAGTGTTAGGAATTTTTTG GCTGGGGCGTTAA BpOF4_13740 ATGAAAGGAAGACCACTTTTACCATTTGCGATCATAGCAATTGT 15 nucleicacidsequence CGGGATTGTTGTTATGATTTCGCTTTCATTTATTGGGTTAAACC AGCGTGAAGCGATGCAGGCAGATGAAGAAGGAGAAGAAGAAGTA ACTGAAATTGAAGATCCGGTAGCAGCTGGAGAAGAATTAGTGCA AACTTCTTGTATCGGTTGTCACGGTGGCGATTTAAGCGGTGGTG CAGGTCCTGCCCTAACGTCTCTTGAAGGTCAATACACTCAAGAA GAAATTACAGATATTGTTGTTAATGGGATTGGATCAATGCCGTC AGTTAACGATAACGAAGTAGAAGCAGACGCAATTGCACAGTATT TACTATCTATTTCAGAATAA BpOF4_13740 MKGRPLLPFAIIAIVGIVVMISLSFIGLNQREAMQADEEGEEEV 16 aminoacidsequence TEIEDPVAAGEELVQTSCIGCHGGDLSGGAGPALTSLEGQYTQE EITDIVVNGIGSMPSVNDNEVEADAIAQYLLSISE*

Example 5: Assay for Lysine Production Potential of Strains Anchoring Individual Genes Thereat

(27) The two strains prepared in Example 4 were cultured in the following manner so as to measure OD values, lysine production yields, and sugar consumption rates (g/hr). First, each strain was inoculated into a 250-ml corner-baffle flask containing 25 ml of a seed medium and then cultured at 30 C. for 20 hours while shaking at 150 rpm. Thereafter, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of a production medium and then cultured at 32 C. for 40 hours while shaking at 150 rpm. Compositions of the seed medium and the production medium were as follow, and the culture results are given in Table 4, below.

(28) <Seed Medium (pH 7.0)>

(29) Glucose 20 g, Peptone 10 g, Yeast extract 5 g, Urea 1.5 g, KH.sub.2PO.sub.44 g, K.sub.2HPO.sub.4 8 g, MgSO.sub.4.7H.sub.2O 0.5 g, Biotin 100 g, Thiamine HCl 1,000 g, Calcium pantothenate 2,000 g, Nicotinamide 2000 g (per liter of distilled water)

(30) <Production Medium (pH 7.0)>

(31) Glucose 45 g, Sugar beet-derived molasses 10 g, Soybean steep liquid 10 g, (NH.sub.4).sub.2SO.sub.4 24 g, MgSO.sub.4.Math.7H.sub.2O 0.6 g, KH.sub.2PO.sub.4 0.55 g, Urea 5.5 g, CaCO.sub.3 30 g, Biotin 0.9 mg, Thiamine HCl 4.5 mg, Calcium pantothenate 4.5 mg, Nicotinamide 30 mg, MnSO.sub.4.Math.5H.sub.2O 9 mg, ZnSO.sub.4.Math.5H.sub.2O 0.45 mg, CuSO.sub.4.Math.5H.sub.2O 0.45 mg, FeSO.sub.4.Math.5H.sub.2O 9 mg, and Kanamycin 25 mg (per liter of distilled water)

(32) TABLE-US-00004 TABLE 4 OD Values, Lysine Production Potential, and Sugar Consumption Rate of Individual Gene-Enhanced Strain Relative FN (final) Sugar Lysine OD Consumption Production Strain FN Rate (%) Yield (%) KCCM11016P 68.5 99.5 18.9 KCCM11016P_2284 67.8 100 18.7 KCCM11016P_2284::PgapA 68.3 103 18.8 BpOF4_13735 KCCM11016P_2284::PgapA 69.1 131.5 18.6 BpOF4_13740

(33) The strain in which BpOF4_13735, which is one of the two genes procured from the gDNA library, was overexpressed did not exhibit a significantly improved effect in terms of lysine production yield and sugar consumption rate, compared to the parent strain KCCM11016P_2284. In contrast, the KCCM11016P_62284::PgapA BpOF4_13740 strain, although similar to the parent strain KCCM11016P_62284 in the final OD and yield, was found to increase in sugar consumption rate per hour over the middle culturing section (17 to 24 hours) by 31.5% compared to the parent strain.

(34) Finally, it was found that the effects of the colonies LYS_Lib.Bps #257, #881, and #4213 revealed in Example 2 were attributed to the enhancement of BpOF4_13740.

Example 6: Enhancement of Lysine Production Potential in BpOF4_13740-Enhanced Strain

(35) In order to secondarily verify the effect of BpOF4_13740 gene confirmed in Example 5, BpOF4_13740 gene was assayed after being enhanced with a different promoter. And the effect was also assayed for the BpOF4_05495 gene further confirmed by NCBI BLAST analysis as same functional protein.

(36) To this end, a gene expression vector was additionally constructed in the same manner as for the vector constructed in Example 3.

(37) A sigB promoter was amplified by PCR using the primers of SEQ ID NOS: 17 and 18, with ATCC13032 gDNA serving as a template. Using the primers of SEQ ID NOS: 19 and 12, PCR was performed on the template of Bacillus pseudofirmus OF4 gDNA to amplify BpOF4_13740 gene. These two gene fragments were ligated to pDZ_2284 to construct pDZ_2284::PsigB BpOF4_13740 vector.

(38) Enhancement of the BpOF4_05495 gene was also achieved in the same manner to construct pDZ_2284::PsigB BpOF4_05495 and pDZ_2284::PgapA BpOF4_05495 vectors. A BpOF4_05495 gene fragment was procured using the primers of SEQ ID NOS: 20 and 21 and the primers of SEQ ID NOS: 22 and 21. An effect obtained upon simultaneous enrichment of the two genes was also examined. In this regard, primers of SEQ ID NOS: 23 and 24, which were designed to contain a ribosome-binding sequence (RBS), were additionally synthesized to construct pDZ_2284::PsigB BpOF4_13740_05495 and pDZ_2284::PgapA BpOF4_13740_05495 vectors.

(39) The five additional vector constructs (pDZ_2284::PsigB BpOF4_13740 vector, pDZ_A2284::PsigB BpOF4_05495 vector, pDZ_2284::PgapA BpOF4_05495 vector, pDZ_2284::PsigB BpOF4_13740_05495 vector, and pDZ_2284::PgapA BpOF4_13740_05495 vector) were each transformed into the lysine producing strain Corynebacterium glutamicum KCCM11016P (Korean Patent No. 10-0159812) by an electric pulse method and subjected to secondary DNA-crossover to prepare strains having the individual genes enhanced therein.

(40) The five strains thus obtained were named KCCM11016P_2284::PsigB BpOF4_13740, KCCM11016P_2284::PsigB BpOF4_05495, KCCM11016P_2284::PgapA BpOF4_05495, KCCM11016P_2284::PsigB BpOF4_13740_05495, and KCCM11016P_2284::PgapA BpOF4_13740_05495, respectively.

(41) The primers, promoters, nucleic acid sequences of BpOF4_05495, and amino acid sequence encoded by the gene used herein are summarized in Table 5, below:

(42) TABLE-US-00005 TABLE4 Description sequence(5.fwdarw.3orN.fwdarw.C) SEQIDNO FprimerforsigB ATTGATGTGGATTTCTGcatTGCAGCACCTGGTGAGGTGG 17 promoter RprimerforsigB AACTGGCCTCCTAAATTCGCGGTTC 18 promoter Fprimerfor GCGAATTTAGGAGGCCAGTTATGAAAGGAAGACCACTTTT 19 BPOF4_13740 Fprimerfor GCGAATTTAGGAGGCCAGTTATGAAAAAGTTTTTATTAGC 20 BpOF4_05495 Rprimerfor TTGGTCAAACCTCCCCTcatTTATTGAGCTTCAAGCCATG 21 BpOF4_05495 Fprimerfor ATCTTTAGAGGAGACACAACATGAAAAAGTTTTTATTAGC 22 BpOF4_05495 RprimerforRBS CTGTGTTTCCTCCTTTCTCCTGTTATTCTGAAATAGATAGTA 23 insertion FprimerforRBS CAGGAGAAAGGAGGAAACACAGATGAAAAAGTTTTTATTAGC 24 insertion sigBpromoter TGCAGCACCTGGTGAGGTGGCTGAGCCGGTGATTGAAAAGATTGCAC 25 AAGGTTTACGTGAGCGCGGAATCACCGTGGAACAAGGACGATTCGGC GCAATGATGAAGGTCACATCGGTTAACGAAGGCCCCTTCACCGTTTT GGTCGAGTGCTAGCCAGTCAATCCTAAGAGCTTGAAACGCCCCAATG TGGGGGTGTTAAGAACTCCATAAAAGCGCTTGGGAACTTTTTGTGGA AGCAGTCCGTTGAACCTCTTGAACCGCGAATTTAGGAGGCCAGTT BPOF4_05495 ATGAAAAAGTTTTTATTAGCTCTTGGCGCAGTTGTTGCTCTTACAGC 26 nucleicacidsequence ATGTGGCGGCGGAGACGAAGCTGCTCCACCGGTTGATGAGGAGTCTC CAGCAGTAGATGAAGCTCCAGCAGATGAGCCTGCAGATGATGCAACA GCTGGTGATTACGATGCAGAATCAGCTCGTGCTACATATGAGCAAAG CTGTATCGCATGTCATGGCGGCGATCTTCAAGGGGCATCAGGTCCAG CTCTAGTAGGAACTGGCCTGTCAGCTGCTGAAATTCAAGACATCATC CAAAACGGACAAGGTTCAATGCCTGCTCAAAATTTAGATGATGACGA AGCTGCTAACCTAGCTGCATGGCTTGAAGCTCAATAA BPOF4_05495 MKKFLLALGAVVALTACGGGDEAPPVDEESPAVDEAPADEPADDATA 27 aminoacidsequence GDYDAESARATYEQSCIACHGGDLQGASGPALVGTGLSAAEIQDIIQ NGQGSMPAQNLDDDEAANLAAWLEAQ

(43) The five strains, KCCM11016P_62284::PsigB BpOF4_13740, KCCM11016P_2284::PsigB BpOF4_05495, KCCM11016P_2284::PgapA BpOF4_05495, KCCM11016P_2284::PsigB BpOF4_13740_05495, and KCCM11016P_2284::PgapA BpOF4_13740_05495, were evaluated in flasks in the same condition as in Example 5 and the results are given in Table 6, below.

(44) TABLE-US-00006 TABLE 6 OD Values, Lysine Production Potentials, and Sugar Consumption Rates in Strains Enhanced with Genes Individually or in Combination FN Relative Lysine FN Sugar OD Conc. Lysine Consumption Strain FN (g/L) Yield % Rate (%) KCCM11016P 68.4 9.0 18.4 101.6 KCCM11016P_2284 68.1 8.7 18.7 100 KCCM11016P_2284::PsigB 67.5 8.4 18.1 107.1 BpOF4_13740 KCCM11016P_2284::PgapA 68.8 9.2 18.4 142.1 BpOF4_13740 KCCM11016P_2284::PsigB 68.8 8.7 18.2 120.2 BpOF4_05495 KCCM11016P_2284::PgapA 68.9 8.9 17.8 136.6 BpOF4_05495 KCCM11016P_2284::PsigB 68.4 9.3 19.3 114.8 BpOF4_13740_05495 KCCM11016P_2284::PgapA 69 9.5 19.0 145.9 BpOF4_13740_05495

(45) Expression of BpOF4_13740 gene under the control of sigB promoter and gapA promoter resulted in increasing sugar consumption rates per hour by 7.1% and 42.1%, respectively, compared to the control KCCM11016P_2284. In addition, when BpOF4_05495 gene, which encodes a similar protein, was additionally introduced under the control of sigB and gapA promoters, the sugar consumption rates increased by 20.2% and 36.6%, respectively. The two results indicate that sugar consumption rates (g/hr) increase with the enhancement of the gene under the control of a promoter. In addition, the sugar consumption rate was observed to peak upon simultaneous expression of the genes BpOF4_13740 and BpOF4_05495. In detail, the strain KCCM11016P_2284::PgapA BpOF4_13740_05495 exhibited a sugar consumption rate per hour increased by 45.9%, compared to the control KCCM11016P_2284.

(46) The strain KCCM11016P_2284::PgapA BpOF4_13740_05495 (called as Corynebacterium glutamicum CM03-885), which has an enhanced lysine production potential, was deposited in the Korean Culture Center of Microorganisms located in Hongje-dong, Seodamun-Gu, Seoul, Korea on Dec. 13, 2019 and given the accession number KCCM 12640P.

Example 7: Assay for Lysine Production Potential of BpOF4_13740_05495-Enhance Strain

(47) Corynebacterium glutamicum KCCM10770P (Korean Patent No. 10-0924065) and KCCM11347P (Korean Patent No. 10-0073610), both of which produce L-lysine, were each enhanced with the genes selected in Example 6. To this end, the genes were introduced in the same manner as in Example 6. Finally, the three vectors pDZ_2284, pDZ_2284::PsigB BpOF4_13740_05495, and pDZ_2284::PgapA BpOF4_13740_05495 were each transformed into the two strains Corynebacterium glutamicum KCCM10770P and KCCM11347P to prepare a total of six strains KCCM10770P_2284, KCCM10770P_2284::PsigB BpOF4_13740_05495, KCCM10770P_2284::PgapA BpOF4_13740_05495, KCCM11347P_2284, KCCM11347P_2284::PsigB BpOF4_13740_05495, and KCCM11347P_2284::PgapA BpOF4_13740_05495.

(48) The gene-enhanced strains thus obtained were cultured in the same manner as in Example 5 and measured for OD, lysine production yield, and relative sugar consumption rate per hour (when the sugar consumption rates per hour of KCCM10770P_2284 and KCCM11347P_2284 were set 100%), and the results are given in Table 7, below.

(49) TABLE-US-00007 TABLE 7 OD Values, lysine production potential, and Relative Sugar Consumption Rate of Gene-Enhanced Strain FN Relative Lysine FN Sugar OD Conc. Lysine Consumption Strain FN (g/L) Yield % Rate (%) KCCM10770P 95.5 6.7 13.3 99.4 KCCM10770P_2284 95.3 6.5 13.0 100 KCCM10770P_2284::PsigB 95.0 6.5 13.0 102.5 BpOF4_13740_05495 KCCM10770P_2284::PgapA 95.9 6.3 12.7 105.6 BpOF4_13740_05495 KCCM11347P 65.0 15.1 30.2 99.4 KCCM11347P_2284 64.7 15.3 30.6 100 KCCM11347P_2284::PsigB 65.5 15.1 30.2 103.5 BpOF4_13740_05495 KCCM11347P_2284::PgapA 65.2 15.5 31.0 108.2 BpOF4_13740_05495

(50) As shown in Table 7, although similar to the control in terms of OD, FN lysine concentration, and lysine production yield, the gene-enhanced strains prepared in this Example were fermented within a shorter time due to the improved sugar consumption rate thereof.

Example 8: Preparation of BpOF4_13740_05495-Introduced CJ3P Strain and Assay for Lysine Production Potential Thereof

(51) An examination was made to see whether different Corynebacterium glutamicum variants producing L-lysine exhibited the same effect as in the foregoing. In this regard, Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13:R40), which is made to have an L-lysine production potential by introducing three mutations [pyc(P458S), hom(V59A), and lysC(T3111)] to the wild-type, was enhance with BpOF4_13740_05495 in the same manner as in Example 7. The enhanced strains thus obtained were named CJ3_2284, CJ3_2284::PsigB BpOF4_13740_05495, and CJ3_2284::PgapA BpOF4_13740_05495, respectively. The control CJ3P strain (not enhanced with BpOF4_13740_05495) and the three prepared strains were cultured in the same manner as in Example 5 and measured for OD, lysine production yield, and relative sugar consumption rate per hour (when the sugar consumption rate per hour in each of KCCM10770P_2284 and KCCM11347P_2284 was set 100%), and the results are given in Table 8, below:

(52) TABLE-US-00008 TABLE 8 OD Values, Lysine Production Potential, and Relative Sugar Consumption Rate in Gene-Enhanced Strains FN Relative Lysine FN Sugar OD Conc. Lysine Consumption Strain FN (g/L) Yield % Rate (%) CJ3 70.5 4.5 9.0 100.4 CJ3_2284 71.4 4.2 8.4 100 CJ3_2284::PsigB 70.9 4.4 8.8 102.9 BpOF4_13740_05495 CJ3_2284::PgapA 71.6 4.6 9.2 160.9 BpOF4_13740_05495

(53) As shown in Table 8, the BpOF4_13740_05495-enhanced strain, although similar to the control in terms of OD, FN lysine concentration, and lysine production yield, was found to increase in sugar consumption rate per hour by 60% or more.

(54) 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 claims and from their equivalents rather than the detailed description.