MUTANT OF CORYNEBACTERIUM GLUTAMICUM WITH ENHANCED L-LYSINE PRODUCTIVITY AND METHOD FOR PREPARING 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 mutant strain may produce L-lysine in an improved yield compared to the parent strain by increasing or enhancing the expression of a gene encoding aspartate-semialdehyde dehydrogenase therein.

Claims

1. A Corynebacterium glutamicum mutant strain having enhanced L-lysine productivity due to an enhanced activity of aspartate-semialdehyde dehydrogenase.

2. The Corynebacterium glutamicum mutant strain of claim 1, wherein the enhanced activity of the aspartate-semialdehyde dehydrogenase is achieved by site-directed mutagenesis of a gene encoding the aspartate-semialdehyde dehydrogenase.

3. The Corynebacterium glutamicum mutant strain of claim 2, wherein the gene encoding the aspartate-semialdehyde dehydrogenase is represented by the amino acid sequence of SEQ ID NO: 1.

4. The Corynebacterium glutamicum mutant strain of claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 3 or 5.

5. A method for producing L-lysine, the method comprising steps of: a) culturing the 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows the structure of a pCGI (asd A35G/S39E/A78V) vector containing an asd gene obtained by alanine-to-glycine substitution at amino acid position 35, serine-to-glutamic acid substitution at amino acid position 39, and alanine-to-valine substitution at amino acid position 78 in the amino acid sequence of the asd gene according to one example of the present disclosure.

[0045] FIG. 2 shows the structure of a pCGI (asd A35G/R38L/S39E/A78V) vector containing an asd gene obtained by alanine-to-glycine substitution at amino acid position 35, arginine-to-leucine substitution at amino acid position 38, serine-to-glutamic acid substitution at amino acid position 39, and alanine-to valine substitution at amino acid position 78 in the amino acid sequence of the asd gene according to one example of the present disclosure.

DETAILED DESCRIPTION

[0046] 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

[0047] To construct a Corynebacterium glutamicum mutant strain having an enhanced activity of aspartate-semialdehyde dehydrogenase, a Corynebacterium glutamicum DS1 strain and E. coli DH5a (HIT Competent cells?, Cat No. RH618) were used.

[0048] The Corynebacterium glutamicum DS1 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.

[0049] 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.

[0050] The antibiotics kanamycin and streptomycin used were purchased from Sigma, and DNA sequencing was performed by Macrogen.

1-1. Construction of Recombinant Vector

[0051] In order to increase lysine productivity by enhancing the activity of aspartate-semialdehyde dehydrogenase involved in the lysine biosynthesis pathway in the strain, the enhancement of the aspartate-semialdehyde dehydrogenase was introduced. In the method used in this Example, specific mutations in the asd gene were induced in order to increase the expression of the asd gene encoding aspartate-semialdehyde dehydrogenase. Alanine-to-glycine substitution at amino acid position 35, serine-to-glutamic acid substitution at amino acid position 39, and alanine-to-valine substitution at amino acid position 78 in the amino acid sequence of the asd gene were performed, and a 1000-bp region of the left arm and a 1785-bp region of the right arm with respect to the region including 35.sup.th, 39.sup.th and 78.sup.th amino acids of the asd 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 (asdA35G/S39E/A78V) (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 TABLE1 SEQID Primer NO Primersfor asd-LA-F1 5-getctcaggctggtgtgct-3 7 amplification asd-LA-F2 5-gaccatgattacgccgctctcaggct 8 ofleft ggtgtgct-3 homologyarm asd-LA-R1 5-GGGAACCAAAGAAACGAACA-3 9 ofasd asd-LA-R2 5-GAAACGAACAGTGTCAGCTG-3 10 Primersfor asd-RA-F1 5-CCACTGTTCGCTGCTGCAGG-3 11 amplification asd-RA-F2 5-GCAGTACGCTCCACTGTTCGCTGCTG 12 ofright CAGG-3 homologyarm asd-RA-R1 5-gatagccgacccgatggccct-3 13 ofasd asd-RA-R2 5-ggcccttattcttggaatca-3 14

[0052] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, TAKARA BIO Inc., Japan), a reaction solution containing 100 ?M of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 1 pM of oligonucleotide, and 10 ng of the chromosomal DNA of the Corynebacterium glutamicum DS1 strain as a template, PCR was performed for 30 cycles in the presence of 1 unit of a pfu-x DNA polymerase mixture (Solgent). The PCR was performed for 30 cycles, each consisting 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).

[0053] 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 ?g/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.

[0054] As the process commonly performed in the above method, the genes of interest was amplified from the genomic DNA of Corynebacterium glutamicum ATCC 13032 genomic DNA 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 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

[0055] A DS4 strain, a mutant strain, was constructed using the pCGI (asd A35G/S39E/A78V) vector. The vector was prepared at a final concentration of 1 ?g/?l 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 ?g/?l 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 of the isolated strain, the isolated strain was inoculated on a CM-agar liquid medium containing streptomycin, cultured overnight, 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 mutations were introduced into the asd 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 (DS4) having the mutant asd gene introduced therein was obtained.

Example 2. Construction of Corynebacterium glutamicum Mutant Strain

[0056] A Corynebacterium glutamicum mutant strain was constructed in the same manner as in Example 1, except that alanine-to-glycine substitution at amino acid position 35, arginine-to-leucine substitution at amino acid position 38, serine-to-glutamic acid substitution at amino acid position 39, and alanine-to valine substitution at amino acid position 78 in the amino acid sequence of the asd gene were performed.

[0057] In this Example, for construction of a plasmid, the primers shown in Table 1 above were used to amplify each gene fragment. A DS4-1 strain, a mutant strain, was constructed using the constructed plasmid pCGI (asd A35G/R38L/S39E/A78V) vector (see FIG. 2). Finally, a Corynebacterium glutamicum mutant strain (DS4-1) having the mutant asd gene introduced therein was obtained.

Experimental Example 1. Comparison of L-Lysine Productivity between Mutant Strains

[0058] L-lysine productivity was compared between the parent strain Corynebacterium glutamicum DS1 strain and the lysine-producing mutant strains DS4 and DS4-1 strains constructed in Examples 1 and 2.

[0059] Each of the strains was inoculated into a 100-ml flask containing 10 ml of a lysine medium having the composition shown in Table 2 below, and then cultured with shaking at 180 rpm at 30? C. for 48 hours. After completion of the culture, the amount of L-lysine produced was measured by HPLC (Shimazu, Japan), and the results of the measurement are shown in Table 3 below.

TABLE-US-00002 TABLE 2 Content Composition (per L of distilled water) Glucose 100 g Ammonium sulfate 55 g (NH.sub.4).sub.2SO.sub.4 35 g KH.sub.2PO.sub.4 1.1 g MgSO.sub.4H.sub.2O 1.2 g MnSO.sub.4H.sub.2O 180 mg FeSO.sub.4H.sub.2O 180 mg ThiamineHCl 9 mg Biotin 1.8 mg CaCO.sub.3 5% pH 7.0

TABLE-US-00003 TABLE 3 L-lysine production L-lysine per gram dry cell weight Strain OD.sub.610 (g/L) (g/gDCW) Parent strain (DS1) 22 64.8 7.0 Mutant strain (DS4) 22.2 71.9 7.7 Mutant strain (DS4-1) 21.6 67.4 7.4

[0060] As shown in Table 3 above, it was confirmed that, in the Corynebacterium glutamicum mutant strains DS4 and DS4-1 in which specific positions (amino acids at positions 35, 39 and 78, or amino acids at positions 35, 38, 39 and 78) in the amino acid sequence of the asd gene were substituted with optimal amino acids to enhance the lysine biosynthesis pathway, the L-lysine productivities of the mutant strains increased by about 10.0% and 5.7%, respectively, compared to that of the parent strain Corynebacterium glutamicum DS1 strain. From these results, it could be seen that enhanced expression of the asd gene enhanced L-lysine productivity of the mutant strain by enhancing the supply of the lysine precursor.

[0061] As described above, the Corynebacterium glutamicum mutant strain according to the present disclosure may produce L-lysine in an improved yield compared to the parent strain by increasing or enhancing the expression of the gene encoding aspartate-semialdehyde dehydrogenase therein.

[0062] 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.