CORYNEBACTERIUM GLUTAMICUM MUTANT STRAIN HAVING ENHANCED L-LYSINE PRODUCTIVITY AND METHOD OF PRODUCING L-LYSINE USING THE SAME

Abstract

The present disclosure relates to a Corynebacterium glutamicum mutant strain having enhanced L-lysine productivity and a method of producing L-lysine using the same. The Corynebacterium glutamicum mutant strain may produce L-lysine in an improved yield by inhibiting the conversion of oxaloacetate to citrate due to decreased or inhibited expression of the gene encoding the citrate synthase.

Claims

1. A Corynebacterium glutamicum mutant strain having enhanced L-lysine productivity due to weakened activity of citrate synthase.

2. The Corynebacterium glutamicum mutant strain of claim 1, wherein the weakened activity of the citrate synthase is achieved by replacement of a start codon of a gene encoding the citrate synthase with GTG.

3. The Corynebacterium glutamicum mutant strain of claim 1, wherein the weakened activity of the citrate synthase is achieved by replacement of a start codon of a gene encoding the citrate synthase with TTG.

4. The Corynebacterium glutamicum mutant strain of claim 2, wherein the gene encoding the citrate synthase is represented by the nucleotide sequence of SEQ ID NO: 1.

5. A method for producing L-lysine, the method comprising steps of: a) culturing the Corynebacterium glutamicum mutant strain of claim 1 in a medium; and b) recovering L-lysine from the mutant strain or the medium in which the mutant strain has been cultured.

6. The Corynebacterium glutamicum mutant strain of claim 3, wherein the gene encoding the citrate synthase is represented by the nucleotide sequence of SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 shows the structure of a pCGI(gltA-A1G) vector containing a citrate synthase (gltA) gene obtained by ATG-to-GTG replacement in the start codon according to one example of the present disclosure.

[0041] FIG. 2 shows the structure of a pCGI(gltA-A1T) vector containing a citrate synthase (gltA) gene obtained by ATG-to-TTG replacement in the start codon according to one example of the present disclosure.

DETAILED DESCRIPTION

[0042] Hereinafter, the present disclosure will be described in more detail. However, this description is provided by way of example only to aid the understanding of the present disclosure, and the scope of the present disclosure is not limited by this illustrative description.

Example 1. Construction of Corynebacterium glutamicum Mutant Strain

[0043] To construct a Corynebacterium glutamicum mutant strain, a Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells™, Cat No. RH618) were used.

[0044] The Corynebacterium glutamicum DS1 strain was cultured in a CM-broth medium (pH 6.8) containing, per liter of distilled water, 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone and 5.0 g of beef extract at a temperature of 30° C.

[0045] The E. coli DH5a was cultured in an LB medium containing, per liter of distilled water, 10.0 g of tryptone, 10.0 g of NaCl and 5.0 g of yeast extract at a temperature of 37° C.

[0046] The antibiotics ampicillin, kanamycin and chloramphenicol used were purchased from Sigma, and DNA sequencing was performed by Macrogen.

1-1. Construction of Recombinant Vector

[0047] In order to weaken the TCA cycle in the strain and increase the carbon source efficiency, weakening of the citrate synthase was introduced into the strain. In the method used in this Example, a specific mutation was induced in the translation start codon of the gltA gene encoding the citrate synthase in order to decrease the expression of the gltA gene. The translation start codon of the gltA gene was mutated from ATG to GTG, and a 478-bp region of the left arm and a 475-bp region of the right arm with respect to the center of the gltA gene on the Corynebacterium glutamicum genome were amplified by PCR, ligated by overlap PCR, and then cloned into the recombinant vector pCGI (see Kim et al., Journal of Microbiological Methods 84 (2011), 128-130). The resulting plasmid was named pCGI(gltA-A1G) (see FIG. 1). For construction of the plasmid, the primers shown in Table 1 below were used to amplify each gene fragment.

TABLE-US-00001 TABLE 1 Primer SEQ ID NO Primers for gltA-LA-F1 5′-tgattacgccggttgcgttatagggtggc-3′ 7 amplification gltA-LA-F2 5′-ggttgcgttatagggtggc-3′ 8 of left gltA-LA-R1 5′-ttgttcggaaaaaaactcttcc-3′ 9 homology arm of A1G-LA-R2 5′-tcaaacacatttgttcggaaa-3′ 10 gltA Primers for A1G-RA-F1 5′-atgtgtttgaaagggatatcgtggctactga-3′ 11 amplification gltA-RA-F2 5′-aagggatatcgtggctactga-3′ 12 of right gltA-RA-R1 5′-agctggtcctggtagtaggtaga-3′ 13 homology arm of gltA-RA-R2 5′-gagtgggttcagctggtcct-3′ 14 gltA

[0048] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, TAKARA BIO Inc., Japan), a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 1 pM of oligonucleotide, and 10 ng of the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, PCR was performed for 25 to 30 cycles in the presence of 1 unit of a pfu-X DNA polymerase mixture (Solgent). The PCR cycles each consisted of (i) denaturation at 94° C. for 30 sec, (ii) annealing at 58° C. for 30 sec, and (iii) extension at 72° C. for 1 to 2 min (a polymerization time of 2 min per kb).

[0049] The gene fragments produced as described above were cloned into the pCGI vector by self-assembly cloning. The vector was transformed into E. coli DH5a, which was then streaked on an LB-agar plate containing 50 μg/ml of kanamycin, and cultured at 37° C. for 24 hours. The finally formed colonies were isolated and whether the inserts would be exactly present in the vector was examined, and then the vector was isolated and used for recombination of the Corynebacterium glutamicum strain.

[0050] As the process commonly performed in the above method, the genes of interest was amplified from the genomic DNA of Corynebacterium glutamicum ATCC 13032 by PCR and inserted into the pCGI vector by self-assembly cloning according to the strategy, followed by selection in E. coli DH5a. For chromosomal base substitution, the gene fragments were amplified individually and ligated by overlap PCR to obtain a target DNA fragment. During genetic manipulation, Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes, and various restriction enzymes and DNA modifying enzymes used were purchased from NEB. These polymerases and enzymes were used according to the supplied buffer and protocols.

1-2. Construction of Mutant Strain

[0051] A DS2 strain, a mutant strain, was constructed using the pCGI(gltA-A1G) vector. The vector was prepared at a final concentration of 1 μg/μl or higher, and introduced into the Corynebacterium glutamicum DS1 strain by electroporation (see Tauch et al., FEMS Microbiology Letters 123 (1994), 343-347), thus inducing primary recombination. At this time, the electroporated strain was plated on a CM-agar plate containing 20 μg/μl of kanamycin, and the colonies were isolated, and then whether the vector would properly inserted into the induced position on the genome was analyzed by PCR and sequencing. In order to induce secondary recombination, the isolated strain was inoculated into a CM-agar liquid medium containing streptomycin, cultured overnight or longer, and then plated on an agar medium containing streptomycin at the same concentration, and the colonies were isolated. Whether the final isolated colonies would have resistance to kanamycin was examined, and then whether mutation was introduced into the gltA gene in the strains having no antibiotic resistance was analyzed by sequencing (see Schafer et al., Gene 145 (1994), 69-73). Finally, a Corynebacterium glutamicum mutant strain (DS2) having the mutant gltA gene introduced therein was obtained.

Example 2. Construction of Corynebacterium glutamicum Mutant Strain

[0052] A Corynebacterium glutamicum mutant strain was constructed in the same manner as in Example 1, except that the start codon of the gltA gene was replaced with TTG.

[0053] In this Example, for construction of a plasmid, the primers shown in Table 2 below were used to amplify each gene fragment. A DS2-1 strain, a mutant strain, was constructed using the constructed plasmid pCGI(gltA-A1T) vector. Finally, a Corynebacterium glutamicum mutant strain (DS2-1) having the mutant gltA gene introduced therein was obtained.

TABLE-US-00002 TABLE 2 Primer SEQ ID NO Primers for gltA-LA-F1 5′-tgattacgccggttgcgttatagggtggc-3′ 15 amplification gltA-LA-F2 5′-ggttgcgttatagggtggc-3′ 16 of left gltA-LA-R1 5′-tcaaacaaatttgttcggaaa-3′ 17 homology arm of A1T-LA-R2 5′-atttgtttgaaagggatatcgtggctactga-3′ 18 gltA Primers for A1T-RA-F1 5′-atgtgtttgaaagggatatcgtggctactga-3′ 19 amplification gltA-RA-F2 5′-aagggatatcgtggctactga-3′ 20 of right gltA-RA-R1 5′-agctggtcctggtagtaggtaga-3′ 21 homology arm of gltA-RA-R2 5′-gagtgggttcagctggtcct-3′ 22 gltA

Experimental Example 1. Comparison of L-Glutamic Acid Productivity Between Mutant Strains

[0054] L-lysine productivity was compared between the parent strain Corynebacterium glutamicum DS1 strain and the lysine-producing mutant strains DS2 and DS2-1 strains constructed in Examples 1 and 2.

[0055] The parent strain (DS1) or the mutant strain (DS2 or DS2-1) was inoculated into a 100-ml flask containing 10 ml of a lysine medium having the composition shown in Table 3 below, and then cultured with shaking at 180 rpm at 30° C. for 28 hours. After completion of the culture, the amount of L-lysine produced was measured by HPLC (Shimadzu, Japan), and the results of the measurement are shown in Table 4 below.

TABLE-US-00003 TABLE 3 Content (per L of Composition distilled water) Glucose 100 g Ammonium sulfate 55 g KH.sub.2PO.sub.4 1.1 g MgSO.sub.4•H.sub.2O 1.2 9 MnSO.sub.4•H.sub.2O 180 mg FeSO.sub.4•H.sub.2O 180 mg Thiamine•HCl 9 mg Biotin 1.8 mg CaCO.sub.3 5% pH 7.0

TABLE-US-00004 TABLE 4 L-lysine production per gram Strain L-lysine (g/L) dry cell weight (g/gDCW) Parent strain (DS1) 65.2 7.0 Mutant strain (DS2) 69.7 7.2 Mutant strain (DS2-1) 69.8 7.2

[0056] As shown in Table 4 above, it was confirmed that, in the Corynebacterium glutamicum mutant strains DS2 and DS2-1 in which the start codon of the gltA gene was replaced with the optimal translation start sequence (GTG or TTG) to improve the lysine biosynthesis pathway, the L-lysine productivities of the mutant strains increased by about 6.9% compared to that of the parent strain Corynebacterium glutamicum DS1 strain.

[0057] From these results, it could be seen that weakened expression of the gltA gene enhanced L-lysine productivity of the mutant strain by decreasing the metabolic flux of carbon sources.

[0058] As described above, the Corynebacterium glutamicum mutant strain according to the present disclosure may produce L-lysine in an improved yield by inhibiting the conversion of oxaloacetate to citrate due to decreased or inhibited expression of the gene encoding the citrate synthase.

[0059] So far, the present disclosure has been described with reference to the embodiments thereof. Those of ordinary skill in the art to which the present disclosure pertains will appreciate that the present disclosure may be embodied in modified forms without departing from the essential characteristics of the present disclosure. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present disclosure is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present disclosure.