Recombinant strain producing L-lysine and construction methods therefor and use thereof
Provided are a method for introducing point mutations to the coding sequence of NCg12176 gene or improving the expression thereof in Corynebacterium glutamicum , and a method for performing point mutations on the promoter region sequence of dapB gene in Corynebacterium glutamicum . The fermentation yield of L-lysine produced by a strain with the mutations can be increased by means of the methods.
1 . A method for producing L-lysine, comprising culturing a first recombinant strain contains a polynucleotide encoding a polypeptide that comprise an amino acid sequence of SEQ ID NO: 3, wherein the lysine residue at position 176 is substituted with an asparagine residue;
the polynucleotide sequence is formed by mutation of the base at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1; and the mutation includes base mutation of adenine (A) to cytosine (C) at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1;
or a second recombinant strain contains the promoter nucleotide sequence comprises the nucleotide sequence obtained by mutating the bases at positions −49, −51 and −54 to −58 in the promoter region shown in SEQ ID NO: 29;
or an expression cassette comprising the promoter nucleotide sequence comprises the nucleotide sequence obtained by mutating the bases at positions −49, −51 and −54 to −58 in the promoter region shown in SEQ ID NO: 29;
or a second recombinant vector comprises the promoter nucleotide sequence comprises the nucleotide sequence obtained by mutating the bases at positions −49, −51 and −54 to −58 in the promoter region shown in SEQ ID NO: 29;
wherein in the second recombinant strain or in the second recombinant vector or in the expression cassette, the nucleotide at position −49 of the promoter region shown in SEQ ID NO: 29 is mutated from cytosine (C) to adenine (A), the nucleotide at position −51 is mutated from guanine (G) to thymine (T), and the nucleotide sequence from positions −54 to −58 is mutated from CTGCA to GGTGT;
and
both the first recombinant strain and the second recombinant strain use Corynebacterium glutamicum YP97158 as the host strain, and recovering L-lysine from the culture.
2 . The method according to claim 1 , wherein, the polynucleotide sequence comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 or the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO: 2.
3 . The method according to claim 1 , wherein in the expression cassette, a coding sequence operably linked to the promoter, or a coding sequence is the coding sequence of dapB gene.
4 . The method according to claim 1 , wherein the second recombinant vector is constructed by ligating the promoter nucleotide sequence with pK18mobsacB plasmid.