Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.
L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for breeding L-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as L-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by L-cysteine. We also found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes L-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more L-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells.
Yamada
Satoshi
S
Department of Bioscience, Fukui Prefectural University, Fukui, Japan.
Awano
Naoki
N
Inubushi
Kyoko
K
Maeda
Eri
E
Nakamori
Shigeru
S
Nishino
Kunihiko
K
Yamaguchi
Akihito
A
Takagi
Hiroshi
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D003470:Culture Media; D003545:Cysteine / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013752:Tetracycline / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003470:Culture Media; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C411954:eamA protein, E coli; D013752:Tetracycline; C024504:bicozamycin; D003545:Cysteine