Overproduction of l -Cysteine and l -Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

Abstract

Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production

“…Plasmid pVK7 was constructed by combining HindIII-digested pAM330 [5], cryptic plasmid isolated from B. lactofermentum ATCC13869, with AvaII-digested pHSG299 (Takara Shuzo) by blunt-end ligation. Plasmids pCE harboring the wild-type cysE gene [2] and pCEM256I harboring the Met256Ile mutant cysE gene [2] were digested with EcoRI. The resultant 1.2-kb EcoRI fragment including the promoter and terminator regions was then ligated into the EcoRI site of pVK7 to construct pVK7-CE or pVK-CEM256I.…”

“…When a substrate other than cysteine was tested, the keto-acid formed was measured using 2,4dinitrophenylhydrazine [9]. SAT activity was measured as described previously [2].…”

“…In order to overproduce cysteine by microorganisms, stable expression of feedback inhibition-desensitized SAT is necessary [2]. However, the gene encoding SAT in C. glutamicum has not yet been identi¢ed, and feedback inhibition-desensitized SAT of C. glutamicum has not yet been reported.…”

“…In order to obtain L-cysteine producers, we previously constructed Escherichia coli cysE gene encoding altered SAT. This gene was genetically desensitized to the feedback inhibition induced by L-cysteine using site-directed mutagenesis to replace the 256th methionine residue [2]. We found that, in the recombinant E. coli cells expressing the altered cysE gene, there was a marked production of Lcysteine plus L-cystine, and that stable expression of feed back inhibition-insensitive SAT was necessary for overproduction of L-cysteine.…”

“…We found that, in the recombinant E. coli cells expressing the altered cysE gene, there was a marked production of Lcysteine plus L-cystine, and that stable expression of feed back inhibition-insensitive SAT was necessary for overproduction of L-cysteine. We have also reported the impor-tance of L-cysteine desulfhydrase (CD) activity of the E. coli host cells [2]. In order to further improve L-cysteine production, a host strain having a lower level of CD activity must be isolated ( Fig.…”