Microorganism of the genus <i>Corynebacterium </i>producing l-lysine and a method for producing l-lysine using the same

Abstract

The present disclosure relates to a microorganism producing L-lysine, and a method for producing L-lysine using the same.

Claims

1. An L-lysine producing microorganism of the genus Corynebacterium, having an improved activity of a protein consisting of the amino acid sequence of SEQ ID NO: 1 compared to its endogenous activity, wherein the improved activity is selected from the group consisting of: (1) an increased copy number of a polynucleotide encoding SEQ ID NO: 1; (2) increased expression of SEQ ID NO: 1 by modification of an expression regulatory sequence, (3) on a chromosome, modification of the expression regulatory sequence or introduction of an alternative expression regulatory sequence so that protein activity of SEQ ID NO: 1 is enhanced compared to prior to the modification, (4) codon optimization of the polynucleotide encoding SEQ ID NO: 1, and (5) combinations thereof; and wherein the improved activity of said SEQ ID NO: 1 results in increased L-lysine production in said Corynebacterium compared to an L-lysine producing Corynebacterium that is not modified to have said improved activity.

2. The L-lysine producing microorganism of the genus Corynebacterium according to claim 1, wherein the microorganism is Corynebacterium glutamicum.

3. A method for producing L-lysine, comprising: culturing an L-lysine producing microorganism of the genus Corynebacterium, having an improved activity of a protein consisting of the amino acid sequence of SEQ ID NO: 1 compared to its endogenous activity, in a medium; and recovering L-lysine from the cultured microorganism or medium thereof, wherein the improved activity is selected from the group consisting of: (1) an increased copy number of a polynucleotide encoding SEQ ID NO: 1; (2) increased expression of SEQ ID NO: 1 by modification of an expression regulatory sequence, (3) on a chromosome, modification of the expression regulatory sequence or introduction of an alternative expression regulatory sequence so that protein activity of SEQ ID NO: 1 is enhanced compared to prior to the modification, (4) codon optimization of the polynucleotide encoding SEQ ID NO: 1, and (5) combinations thereof; and wherein the improved activity of said SEQ ID NO: 1 results in increased L-lysine production in said Corynebacterium compared to an L-lysine producing Corynebacterium that is not modified to have said improved activity.

4. The method for producing L-lysine according to claim 3, wherein the microorganism is Corynebacterium glutamicum.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Hereinafter, the present disclosure will be described in detail through exemplary embodiments. However, these exemplary embodiments are provided for the purpose of illustration only, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1: Preparation of Wild-Type Library of Microorganism of Genus Corynebacterium

(2) Upon extracting genomic DNA from Corynebacterium glutamicum ATCC13032, the genomic DNA was treated with Sau3AI restriction enzyme and the DNA fragments were separated by size through electrophoresis on agarose gel, and thereby DNA fragments of 3 kb to 4 kb were selectively obtained. After ligation of the fragments with pECCG117 vector (Korean Patent No. 10-0057684), which has a BamHI restriction site, and introduction into E. coli DH5, the resultants were then plated on solid LB medium containing kanamycin (25 mg/L), and thus transformed colonies were obtained. PCR was performed on 100 random colonies using primers of SEQ ID NOs: 3 and 4, and thereby it was found that the ratio of the colonies containing the vector in which the target DNA fragments of approximately 3 kb to 4 kb were inserted was 90% or higher. All of the obtained colonies were co-cultured after inoculation in kanamycin (25 mg/L)-containing liquid LB medium; plasmids were then extracted using a commonly known plasmid extraction method, thereby completing the Corynebacterium glutamicum ATCC13032 genomic DNA library.

(3) TABLE-US-00001 SEQIDNO:3: 5-ACGACGGGATCAGTACCGA-3 SEQIDNO:4: 5-AGCTATCTGTCGCAGCGCC-3

Example 2: Preparation and Evaluation of Lysine-Producing Microorganism Introduced with Wild-Type Library

(4) Using an electric pulse method, the genomic DNA library prepared in Example 1 was introduced into the lysine-producing strain Corynebacterium glutamicum KCCM11016P (originally designated as KFCC10881, the microorganism was re-deposited at an international depositary institution under the Budapest Treaty and designated as Accession No. KCCM11016P; Korean Patent No. 10-0159812), and after plating on a complex plate medium containing kanamycin (25 mg/L) and culturing at 30 C. for 24 hours, approximately 2,000 colonies were obtained.

(5) <Complex Plate Medium>

(6) 20 g glucose, 50 g (NH.sub.4).sub.2SO.sub.4, 10 g peptone, 5 g yeast extract, 1.5 g urea, 5 g KH.sub.2PO.sub.4, 10 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 g biotin, 1000 g thiamine HCl, 2000 g calcium pantothenate, 2000 g nicotinamide, 20 g agar, 25 mg kanamycin (per 1 L distilled water)

(7) 200 L of a complex liquid medium was dispensed into each well of a 96-well cell culture plate, and after inoculation of each of the obtained colonies, shake-culturing was performed for 24 hours under conditions of 30 C. and 1200 rpm. The cell bodies and supernatant were separated by centrifugation of the culture broth, and 50 L of the supernatant was mixed with a reaction solution containing lysine oxidase.

(8) <Complex Liquid Medium>

(9) 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH.sub.2PO.sub.4, 8 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 g biotin, 1000 g thiamine HCl, 2000 g calcium pantothenate, 2000 g nicotinamide, 25 mg kanamycin (per 1 L distilled water)

(10) <Reaction Solution>

(11) 0.02 units lysine oxidase (Sigma-Aldrich), 0.2 units peroxidase (Sigma-Aldrich), 2 mg ABTS (per 1 mL potassium phosphate buffer solution)

(12) Thereafter, absorbance at OD.sub.405 was analyzed for 30 minutes, and 15 experimental groups exhibiting higher absorbance than the control group (KCCM11016P/pECCG117) were selected. In order to confirm the lysine productivity of each transformant, each strain was inoculated in a 250 mL corner-baffle flask containing 25 mL of a kanamycin (25 mg/L)-containing seed medium and shake-cultured for 20 hours under conditions of 30 C. and 200 rpm. 1 mL of the seed culture broth was inoculated in a 250 mL corner-baffle flask containing 24 mL of a kanamycin (25 mg/L)-containing production medium and shake-cultured for 96 hours at 37 C. and 200 rpm. After termination of the culturing, L-lysine concentration was analyzed using HPLC (Table 1).

(13) <Seed Medium>

(14) 20 g glucose, 5 g (NH.sub.4).sub.2SO.sub.4, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH.sub.2PO.sub.4, 8 g K.sub.2HPO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 g biotin, 1000 g thiamine HCl, 2000 g calcium pantothenate, 2000 g nicotinamide (per 1 L distilled water)

(15) <Production Medium (pH 7.0)>

(16) 100 g glucose, 40 g (NH.sub.4).sub.2SO.sub.4, 2.5 g soybean protein, 5 g corn steep solids, 3 g urea, 1 g KH.sub.2PO.sub.4, 0.5 g MgSO.sub.4.7H.sub.2O, 100 g biotin, 1000 g thiamine HCl, 2000 g calcium pantothenate, 3000 g nicotinamide, 30 g CaCO.sub.3 (per 1 L distilled water)

(17) TABLE-US-00002 TABLE 1 Average Average Lysine lysine Lysine lysine concen- concen- concen- concen- tration tration tration tration Strain No. (g/L) (g/L) Strain No. (g/L) (g/L) KCCM11Q16P/ 1 43.2 43.8 KCCM11016P/M24 1 46.8 46.5 pECCG117 2 44.3 2 46.0 3 43.9 3 46.7 KCCM11016P/A56 1 43.5 43.4 KCCM11016P/O9 1 44.0 43.8 2 44.3 2 43.8 3 42.5 3 43.6 KCCM11016P/C2 1 41.8 42.2 KCCM11016P/O21 1 43.1 42.8 2 42.6 2 42.5 3 42.3 3 42.9 KCCM11016P/D34 1 45.2 44.6 KCCM11016P/P85 1 43.1 43.4 2 44.5 2 43.5 3 44.0 3 43.6 KCCM11016P/F90 1 43.9 44.6 KCCM11016P/P95 1 44.6 44.4 2 45.5 2 44.3 3 44.4 3 44.4 KCCM11016P/H15 1 47.6 46.8 KCCM11016P/R48 1 42.6 42.9 2 46.2 2 42.4 3 46.7 3 43.6 KCCM11016P/J66 1 44.3 43.8 KCCM11016P/S47 1 42.5 41.7 2 43.5 2 41.0 3 43.6 3 41.6 KCCM11016P/K14 1 42.9 43.4 KCCM11016P/U77 1 43.7 43.9 2 43.5 2 44.5 3 43.9 3 43.6

(18) From the above results, KCCM11016P/H15 and KCCM11016P/M24 were selected, which showed an effect of increased lysine productivity compared to the control group, and plasmids were extracted using a commonly known plasmid extraction method. The plasmid derived from KCCM11016P/H15 was named pEC-H15, and that derived from KCCM11016P/M24 was named pEC-M24. Thereafter, nucleotide sequence analysis was carried out using the primers of SEQ ID NOs: 3 and 4. As a result, the pEC-H15 and pEC-M24 plasmids were found to contain the nucleotide sequences of SEQ ID NOs: 15 and 16, respectively. Thus, it was found that both of the above plasmids contain the nucleotide sequence of SEQ ID NO: 2, which encodes the amino acid sequence of SEQ ID NO: 1. Accordingly, the gene encoding the amino acid sequence of SEQ ID NO: 1 was named HM1524, and is hereinafter referred to as such.

Example 3: Preparation of HM1524 Gene Overexpression Vector

(19) In order to confirm the effect of HM1524 found in Example 2, a vector for overexpressing the corresponding gene was prepared.

(20) Based on the reported nucleotide sequences, primers designed to allow insertion of XhoI restriction sites at the 5 and 3 ends (SEQ ID NOs: 5 and 6, respectively) were synthesized in order to obtain the DNA fragment containing the region from about 200 bp upstream of the HM1524 start codon to about 50 bp downstream of the stop codon thereof, and PCR was performed using the genomic DNA of Corynebacterium glutamicum as a template. PCR was performed by initial denaturation at 94 C. for 5 minutes; 30 cycles consisting of denaturation at 94 C. for 30 seconds, annealing at 56 C. for 30 seconds, and polymerization at 72 C. for 90 seconds; and final polymerization at 72 C. for 7 minutes.

(21) TABLE-US-00003 SEQIDNO:5: 5-TCACTCGAGTGATGGCCAGGTTGTTGTC-3 SEQIDNO:6: 5-TCACTCGAGTTAGTCATAGGTACTAGTTT-3

(22) After treatment of the above PCR amplification product with XhoI restriction enzyme, pECCG117 vector was treated with XhoI, ligated with the obtained DNA fragments, and transformed into E. coli DH5, and the resultant was plated on solid LB medium containing kanamycin (25 mg/L). After PCR screening (using SEQ ID NOs: 3 and 4) of the colonies transformed with the vector in which the target gene was inserted, the plasmid was obtained using a commonly known plasmid extraction method, and this plasmid was named pECCG-HM1524.

Example 4: Analysis of Lysine Productivity of Strain Introduced with IIM1524 Gene Overexpression Vector

(23) Upon introduction of the pECCG-HM1524 vector prepared in Example 3 into Corynebacterium glutamicum KCCM11016P (i.e., lysine-producing strain) using an electric pulse method, the resultants were plated on the kanamycin (25 mg/L)-containing complex plate medium, and colonies were obtained after culturing for 24 hours at 30 C. The obtained strain was named KCCM11016P/pECCG-HM1524, and the L-lysine concentration of the culture broth was analyzed after culturing three batches according to the flask culture method of Example 2 (Table 2).

(24) TABLE-US-00004 TABLE 2 Batch 1 Batch 2 Batch 3 Ly- Av- Ly- Av- Ly- Av- sine erage sine erage sine erage con- lysine con- lysine con- lysine cen- con- cen- con- cen- con- tra- cen- tra- cen- tra- cen- tion tration tion tration tion tration Strain No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) KCCM11016P 1 43.9 44.0 43.5 43.9 43.1 43.6 2 44.2 43.7 43.9 3 43.8 44.5 43.8 KCCM11016P/ 1 46.8 46.9 45.6 46.2 46.5 46.3 pECCG- 2 46.5 46.8 45.1 HM1524 3 47.5 46.1 47.2

(25) As a result, it was found that the lysine productivity of KCCM11016P/pECCG-HM1524, the strain in which the HM1524 gene was overexpressed, was increased by 6% compared to that of the parental strain, KCCM11016P.

Example 5: Preparation of Vector for Further Chromosomal Insertion of HM1524 Gene

(26) In order to confirm the effect of the HM1524 gene found in Example 4, a vector was prepared for further insertion of the gene on the chromosome of Corynebacterium. In order to amplify Pcj7 promoter, derived from Corynebacterium ammoniagenes (Korean Patent No. 10-0620092), primers were synthesized which were designed to allow insertion of an EcoRI restriction site at the 5 end and an NdeI restriction site at the 3 end of the Pcj7 promoter (SEQ ID NOs: 7 and 8, respectively), and to allow insertion of a SpeI restriction site at the 5 end and a SalI restriction site at the 3 end of the Pcj7 promoter (SEQ ID NOs: 9 and 10, respectively). As a result of performing PCR using the synthesized primers (SEQ ID NOs: 7 and 8, and SEQ ID NOs: 9 and 10) with the genomic DNA of Corynebacterium ammoniagenes as a template, Pcj7 promoter DNA fragments were obtained containing EcoRI and NdeI restriction sites at the 5 and 3 ends, respectively, as well as SpeI and SalI restriction sites at the 5 and 3 ends, respectively. PCR was performed by initial denaturation at 94 C. for 5 minutes; 30 cycles consisting of denaturation at 94 C. for 30 seconds, annealing at 56 C. for 30 seconds, and polymerization at 72 C. for 30 seconds; and final polymerization at 72 C. for 7 minutes.

(27) Based on the reported nucleotide sequences, primers designed to allow insertion of an NdeI restriction site at the start codon position and a SpeI restriction site downstream of the stop codon (SEQ ID NOs: 11 and 12, respectively) were synthesized in order to amplify the ORF of the HM1524 gene. As a result of performing PCR using the primers of SEQ ID NOs: 11 and 12 with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, HM1524 gene DNA fragments were obtained containing NdeI and SpeI restriction sites at the start codon position and downstream of the stop codon, respectively. PCR was performed by initial denaturation at 94 C. for 5 minutes; 30 cycles consisting of denaturation at 94 C. for 30 seconds, annealing at 56 C. for 30 seconds, and polymerization at 72 C. for 90 seconds; and final polymerization at 72 C. for 7 minutes.

(28) TABLE-US-00005 SEQIDNO:7: 5-TCAGAATTCTTCCTTCAGGCTAATCTTTT-3 SEQIDNO:8: 5-TCACATATGTGTTTCCTTTCGTTGGGTAC-3 SEQIDNO:9: 5-TCAACTAGTCTTCCTTCAGGCTAATCTTT-3 SEQIDNO:10: 5-TCAGTCGACTGTTTCCTTTCGTTGGGTAC-3 SEQIDNO:11: 5-TCACATATGCGCGTAGCTATGATTTC-3 SEQIDNO:12: 5-TCAACTAGTTTAGCCGTGATGCGTTTCAC-3

(29) Upon treatment of each of the above three PCR amplification products with the restriction enzymes corresponding to the restriction sites at each end, pDZ vector (Korean Patent No. 10-0924065) was ligated to the DNA fragments, which were obtained after treatment with EcoR and SalI restriction enzymes, and thereby pDZ-Pcj7-HM1524 vector was prepared.

Example 6: Analysis of Lysine Productivity of Strain with Further Chromosomal Insertion of HM1524 Gene

(30) The pDZ-Pcj7-HM1524 vector prepared in Example 5 was introduced into Corynebacterium glutamicum KCCM11016P using an electric pulse method, and among the colonies transformed by way of homologous recombination, those in which the HM1524 gene was inserted downstream of the HM1524 gene stop codon on the chromosome were selected. The primers of SEQ ID NOs: 13 and 14 were used for screening of the colonies by PCR. The selected strain was named KCCM11016P::Pcj7-HM1524, and the L-lysine concentration of the culture broth was analyzed after culturing according to the flask culture method of Example 2 (Table 3).

(31) TABLE-US-00006 SEQIDNO:13: 5-GTCGAACACGCCAGAACATT-3 SEQIDNO:14: 5-TACTCTCACGATCTCACCCT-3

(32) TABLE-US-00007 TABLE 3 Batch 1 Batch 2 Batch 3 Ly- Av- Ly- Av- Ly- Av- sine erage sine erage sine erage con- lysine con- lysine con- lysine cen- con- cen- con- cen- con- tra- cen- tra- cen- tra- cen- tion tration tion tration tion tration Strain No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) KCCM11016P 1 45.4 44.9 44.5 44.6 44.3 44.5 2 44.8 44.3 45.1 3 44.5 44.9 44.1 KCCM11016P:: 1 47.7 47.4 46.8 47.1 4.72 47.2 pcj-HM1524 2 47.1 47.5 46.9 3 47.5 47.1 47.4

(33) As a result, it was found that the lysine productivity of KCCM11016P::Pcj7-HM1524, the strain in which the HM1524 gene was further inserted, was increased by about 6% compared to that of the parental strain, KCCM11016P. The KCCM11016P::Pcj7-HM1524 strain was named CA01-2297, and was deposited at the Korean Culture Center of Microorganisms (KCCM), an international depositary institution under the Budapest Treaty, on Aug. 2, 2016, being designated as Accession No. KCCM11876P.

Example 7: Production of Lysine Using KCCM10770P-Derived Microorganism in Which HM1524 Gene is Further Inserted

(34) The pDZ-Pcj7-HM1524 vector prepared in Example 5 was transformed into Corynebacterium glutamicum KCCM10770P, a lysine-producing strain (Korean Patent No. 10-0924065). The above Corynebacterium glutamicum KCCM10770P features in that 7 genes associated with the L-lysine biosynthetic pathway are inserted on its chromosome. Colonies were selectively isolated by PCR, and the strain introduced with the HM1524 gene was named Corynebacterium glutamicum KCCM10770P::Pcj7-HM1524. Thereafter, the L-lysine concentration of the culture broth was analyzed after culturing according to the flask culture method of Example 2 (Table 4).

(35) TABLE-US-00008 TABLE 4 Batch 1 Batch 2 Batch 3 Ly- Av- Ly- Av- Ly- Av- sine erage sine erage sine erage con- lysine con- lysine con- lysine cen- con- cen- con- cen- con- tra- cen- tra- cen- tra- cen- tion tration tion tration tion tration Strain No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) KCCM10770P 1 45.7 46.1 45.8 45.9 45.8 45.5 2 46.3 46.2 45.1 3 46.2 45.8 45.7 KCCM10770P:: 1 47.8 48.3 48.5 48.1 47.5 47.7 Pcj-HM1524 2 48.5 47.5 47.5 3 48.7 48.2 48.1

(36) As a result, it was found that the lysine productivity of the strain Corynebacterium glutamicum KCCM10770P::Pcj7-HM1524 was increased by about 5% compared to that of the parental strain.

Example 8: Production of Lysine Using CJ3P-Derived Microorganism in which HM1524 Gene is Further Inserted

(37) The pDZ-Pcj7-HM1524 vector prepared in Example 5 was transformed into Corynebacterium glutamicum CJ3P, a lysine-producing strain (Binder et al. Genome Biology 2012, 13:R40). Corynebacterium glutamicum CJ3P features in that 3 genes associated with the enhancement of L-lysine productivity are inserted on its chromosome. Colonies were selectively isolated by PCR, and the strain introduced with the HM1524 gene was named Corynebacterium glutamicum CJ3P::Pcj7-HM1524. The L-lysine concentration of the culture broth was analyzed after culturing according to the flask culture method of Example 2 (Table 5).

(38) TABLE-US-00009 TABLE 5 Batch 1 Batch 2 Batch 3 Ly- Av- Ly- Av- Ly- Av- sine erage sine erage sine erage con- lysine con- lysine con- lysine cen- concen- cen- concen- cen- concen- tration tration tration tration tration tration Strain No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) CJ3P 1 8.0 8.0 7.5 8.4 8.1 8.1 2 7.5 8.5 7.9 3 8.6 9.1 8.2 CJ3P:: 1 11.5 11.2 10.5 11.4 11.5 11.2 Pcj7- 2 11.2 11.5 11.4 HM1524 3 10.9 12.1 10.6

(39) As a result, it was found that the lysine productivity of the strain Corynebacterium glutamicum CJ3P::Pcj7-HM1524 was increased by about 38% compared to that of the parental strain.

Example 9: Production of Lysine Using KCCM11347P-Derived Microorganism in Which HM1524 Gene is Further Inserted

(40) The pDZ-Pcj7-HM1524 vector prepared in Example 5 was transformed into Corynebacterium glutamicum KCCM11347P, a lysine-producing strain (originally designated as KFCC10750, the microorganism was re-deposited at an international depositary institution under the Budapest Treaty and designated as Accession No. KCCM11347P; Korean Patent No. 10-0073610). Corynebacterium glutamicum KCCM11347P features in that 3 genes associated with the enhancement of L-lysine productivity are inserted on its chromosome. Colonies were selectively isolated by PCR, and the strain introduced with the HM1524 gene was named Corynebacterium glutamicum KCCM11347P::Pcj7-HM1524. The L-lysine concentration of the culture broth was analyzed after culturing according to the flask culture method of Example 2 (Table 6).

(41) TABLE-US-00010 TABLE 6 Batch 1 Batch 2 Batch 3 Av- Av- Av- Ly- erage Ly- erage Ly- erage sine lysine sine lysine sine lysine con- con- con- con- con- con- cen- cen- cen- cen- cen- cen- tra- tration tra- tration tra- tration Strain No. (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) KCCM11347P 1 38.6 38.3 37.5 37.9 37.5 38.0 2 37.8 38.2 38.6 3 38.5 38.0 37.9 KCCM11347P:: 1 41.5 41.9 42.1 41.9 41.6 41.7 Pcj7-HM1524 2 42.0 41.9 41.8 3 42.3 41.6 41.7

(42) As a result, it was found that the lysine productivity of the strain Corynebacterium glutamicum KCCM11347P::Pcj7-HM1524 was increased by about 10% compared to that of the parental strain.

(43) Taken together, the above results demonstrate that for strains having improved activity of the HM1524 gene compared to an endogenous activity thereof, lysine productivity is improved, and further suggest that lysine can be produced in a large quantity by improving the activity of the protein encoded by the above gene in a microorganism.

(44) From the foregoing, those skilled 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.

(45) [Accession No.]

(46) Name of Depositary Institution: Korean Culture Center of Microorganisms (KCCM)

(47) Accession No.: KCCM11876P

(48) Date of Accession: Aug. 2, 2016