OVERPRODUCTION OF L-CYSTEINE AND L-CYSTINE BY ESCHERICHIA-COLI STRAINS WITH A GENETICALLY ALTERED SERINE ACETYLTRANSFERASE

Organisms that overproduced L-cysteine and L-cystine from glucose were constructed by using Escherichia coli K-12 strains, cysE genes coding for altered serine acetyltransferase, which was genetically desensiti zed to feedback inhibition by L-cysteine, were constructed by replacin g the methionine residue at position 256 of the serine acetyltransfera se protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 t hrough site-directed mutagenesis by using PCR, A cysteine auxotroph, s train JM39, was transformed with plasmids having these altered cysE ge nes. The serine acetyltransferase activities of most of the transforma nts, which were selected based on restored cysteine requirements and a mpicillin resistance, were less sensitive than the serine acetyltransf erase activity of the wild type to feedback inhibition by L-cysteine, At the same time, these transformants produced approximately 200 mg of L-cysteine plus L-cystine per liter, whereas these amino acids were n ot detected in the recombinant strain carrying the wild-type serine ac etyltransferase gene. However, the production of L-cysteine and L-cyst ine by the transformants was very unstable, presumably due to a cystei ne-degrading enzyme of the host, such as cysteine desulfhydrase, There fore, mutants that did not utilize cysteine were derived from host str ain JM39 by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Whe n a newly derived host was transformed with plasmids having the altere d cysE genes, we found that the production of L-cysteine plus L-cystin e was markedly increased compared to production in JM39.