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Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans

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Abstract

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.

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Acknowledgments

This work was supported in part by Grant-in-Aid from the Japan Society for the Promotion of Science.

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Author notes

  1. Hirohide Toyama

    Present address: Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa, 903-0213, Japan

Authors and Affiliations

  1. Technical Research Laboratories, Kyowa Hakko Bio Co. Ltd., Yamaguchi, 747-8522, Japan

    Shihomi Nishikura-Imamura & Tetsuya Abe

  2. Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan

    Minenosuke Matsutani, Hirohide Toyama, Toshiharu Yakushi, Osao Adachi & Kazunobu Matsushita

  3. Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Chayatip Insomphun & Alisa S. Vangnai

Authors
  1. Shihomi Nishikura-Imamura
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  2. Minenosuke Matsutani
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  3. Chayatip Insomphun
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  4. Alisa S. Vangnai
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  5. Hirohide Toyama
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  6. Toshiharu Yakushi
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  7. Tetsuya Abe
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  8. Osao Adachi
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  9. Kazunobu Matsushita
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Correspondence to Toshiharu Yakushi.

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Nishikura-Imamura, S., Matsutani, M., Insomphun, C. et al. Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans . Appl Microbiol Biotechnol 98, 2955–2963 (2014). https://doi.org/10.1007/s00253-013-5439-z

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