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 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 of L-cysteine plus L-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetytransferase gene. However, the production of L-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 with N-methyl-N'-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production of L-cysteine plus L-cystine was markedly increased compared to production in JM39.
Nakamori
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S
Department of Bioscience, Fukui Prefectural University, Japan. nakamori@fpu.ac.jp
Kobayashi
S I
SI
Kobayashi
C
C
Takagi
H
H
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000096:biosynthesis*; D003553:Cystine / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D051050:Serine O-Acetyltransferase; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D003553:Cystine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003545:Cysteine