PCR random mutagenesis into Escherichia coli serine acetyltransferase: isolation of the mutant enzymes that cause overproduction of L-cysteine and L-cystine due to the desensitization to feedback inhibition.
PCR random mutagenesis in the cysE gene encoding Escherichia coli serine acetyltransferase was employed to isolate the mutant enzymes that, due to a much less feedback inhibition by L-cysteine, cause overproduction of L-cysteine and L-cystine in the recombinant strains. The L-cysteine auxotrophic and non-utilizing E. coli strain was transformed with plasmids having the altered cysE genes. Then, several transformants overproducing L-cysteine were selected by detecting the halo formation of the L-cysteine auxotroph. The production test of amino acids and analysis of the catalytic property on the mutant enzymes suggest that the carboxy-terminal region of serine acetyltransferase plays an important role in the desensitization to feedback inhibition and the high level production of L-cysteine and L-cystine.
Takagi
H
H
Department of Bioscience, Fukui Prefectural University, Japan. hiro@fpu.ac.jp
Kobayashi
C
C
Kobayashi
S
S
Nakamori
S
S
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D003545:Cysteine / Q000096:biosynthesis*; D003553:Cystine / Q000096:biosynthesis*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005246:Feedback; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051050:Serine O-Acetyltransferase
D017931:DNA Primers; D011994:Recombinant Proteins; D003553:Cystine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003545:Cysteine