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The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli

  • Applied Genetics and Molecular Biotechnology
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Abstract

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.

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Acknowledgments

We greatly appreciate R. Takeuchi, N. Yoshida, and S. Morigasaki (Nara Institute of Science and Technology, Japan) for their helpful assistance and discussion on this work, respectively. We thank N. Tsukagoshi (Tokyo Institute of Technology, Yokohama, Japan), A. Yamaguchi (Osaka University, Osaka, Japan), and Ajinomoto, Co., Inc. (Tokyo, Japan) for providing the strains and plasmids. This work was supported in part by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas “Applied Genomics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Ajinomoto Co., Inc. to H.T.

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  1. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

    Natthawut Wiriyathanawudhiwong, Iwao Ohtsu, Zhao-Di Li, Hirotada Mori & Hiroshi Takagi

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  1. Natthawut Wiriyathanawudhiwong
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  2. Iwao Ohtsu
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  3. Zhao-Di Li
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  4. Hirotada Mori
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  5. Hiroshi Takagi
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Correspondence to Hiroshi Takagi.

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Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work.

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Wiriyathanawudhiwong, N., Ohtsu, I., Li, ZD. et al. The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli . Appl Microbiol Biotechnol 81, 903–913 (2009). https://doi.org/10.1007/s00253-008-1686-9

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