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Applied and Environmental Microbiology, July 2006, p. 4735-4742, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02507-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli

Satoshi Yamada,¹ Naoki Awano,¹ Kyoko Inubushi,¹ Eri Maeda,¹ Shigeru Nakamori,¹ Kunihiko Nishino,² Akihito Yamaguchi,² and Hiroshi Takagi^1*

Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Fukui 910-1195,¹ Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan²

Received 24 October 2005/ Accepted 25 April 2006

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.

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* Corresponding author. Present address: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Phone: 81-743-72-5420. Fax: 81-743-72-5429. E-mail: hiro{at}bs.naist.jp.

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Applied and Environmental Microbiology, July 2006, p. 4735-4742, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02507-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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