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

摘要

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/1 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 #bate#-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-production 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 the revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the L-methionineproducing 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.