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

Abstract

ABSTRACT 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.