The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli.
L-cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of L-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in L-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in L-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to L-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for L-cysteine tolerance in E. coli cells. However, L-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of L-cysteine, the transformants exhibited more L-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in L: -cysteine tolerance probably due to its export ability and that TolC overexpression is effective for L-cysteine production in E. coli.
Wiriyathanawudhiwong
Natthawut
N
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Ohtsu
Iwao
I
Li
Zhao-Di
ZD
Mori
Hirotada
H
Takagi
Hiroshi
H
D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D018272:Porins / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D018272:Porins; C073126:tolC protein, E coli; D003545:Cysteine