Improved fermentative l-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli

Applied Microbiology and Biotechnology volume 101, pages6879–6889(2017)Cite this article

Abstract

Sulfate (SO4 2−) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (S2O3 2−), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-l-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential l-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO3 2−) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2−) → l-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to l-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.

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Acknowledgements

We would like to thank Dr. Takako Hishiki (Clinical and Translational Research Center, Keio University School of Medicine, Tokyo, Japan; Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan) and Dr. Makoto Suematsu (Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan; Japan Science and Technology Agency, Exploratory Research for Advanced Technology (ERATO) Suematsu Gas Biology Project), for providing fundamental method of sulfur metabolomic analysis with LC-MS/MS. We would like to thank Dr. Hirotada Mori (Nara Institute of Science and Technology, Nara, Japan) for providing the strains and plasmids. We would like to thank Taka-Aki Sato (Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Japan; Shimadzu Co., Kyoto, Japan) for excellent discussion. We would like to thank Editage (www.editage.jp) for English language editing. This work was supported in part by JSPS KAKENHI Grant Numbers JP26450091 and JP15KT0028, by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (26027AB) from MAFF, Japan, and by the grant from the SKYLARK Food Science Institute, Japan, to I.O. This work was also supported in part by JSPS KAKENHI Grant Numbers JP16K18675 and JP15KT0028 to Y.K. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Affiliations

  1. Innovation Medical Research Institute, University of Tsukuba, Tsukuba, Japan

    Yusuke Kawano, Maeka Shiroyama, Naoyuki Tanaka, Satoshi Oshiro & Iwao Ohtsu

  2. Research Institute for Bioscience Products and Fine Chemicals, Ajinomoto Co., Kawasaki, Japan

    Fumito Onishi & Gen Nonaka

  3. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan

    Masashi Miura & Iwao Ohtsu

  4. MS Business Unit, Shimadzu Co., Nakagyo, Japan

    Tsuyoshi Nakanishi

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  1. Yusuke Kawano
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  2. Fumito Onishi
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Correspondence to Iwao Ohtsu.

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Kawano, Y., Onishi, F., Shiroyama, M. et al. Improved fermentative l-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli . Appl Microbiol Biotechnol 101, 6879–6889 (2017). https://doi.org/10.1007/s00253-017-8420-4

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Keywords

  • l-Cysteine
  • l-Cysteine production
  • Sulfur assimilation
  • Thiosulfate sulfurtransferase
  • Escherichia coli