Mechanism of L-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of L-methionine biosynthetic enzymes.
We derived L-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selected the potent L-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg L-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The L-methionine biosynthetic enzymes, cystathionine gamma-synthase and cystathionine beta-lyase, of the L-methionine-overproducing mutants were little repressed by L-methionine. To analyse the mechanism of L-methionine overproduction in the mutant strains, the metJ gene coding for the E. coli met repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser-->Asn) at position 54 was observed in four independent L-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the L-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the L-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in L-methionine overproduction, probably because of the derepression of L-methionine biosynthetic enzymes.
Nakamori
S
S
Department of Bioscience, Fukui Prefectural University, Japan.
Kobayashi
S
S
Nishimura
T
T
Takagi
H
H
D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D004345:Drug Industry; D004352:Drug Resistance, Microbial; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007218:Industrial Microbiology; D008715:Methionine / Q000031:analogs & derivatives / Q000096:biosynthesis*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; C489220:methionine repressor protein, E coli; D008715:Methionine