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Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli

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Abstract

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

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Acknowledgments

We thank Dr. Changfu Zhu and Dr. Gerhard Sandmann for giving us the GlZEP cDNA clone. We also thank Dr. Hisashi Harada for giving us the Paeodactylum tricornutum genome DNA. The authentic samples violaxanthin and antheraxanthin are gifts from Dr. Kazutoshi Shindo.

Funding

This study was funded by the “Smart Cell Project” organized by the New Energy and Industrial Technology Development Organization (NEDO) (16100920-0).

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Authors and Affiliations

  1. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836, Japan

    Miho Takemura, Yuki Higuchi, Takashi Maoka & Norihiko Misawa

  2. Institute of Health Sciences, Ezaki Glico Co., Ltd., Osaka, 555-8502, Japan

    Akiko Kubo & Kohji Ohdan

  3. Division of Food Function and Chemistry, Research Institute for Production Development, Kyoto, 606-0805, Japan

    Takashi Maoka

  4. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8566, Japan

    Takehiko Sahara & Katsuro Yaoi

  5. Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, 263-8522, Japan

    Daisuke Umeno

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  1. Miho Takemura
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  2. Akiko Kubo
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  3. Yuki Higuchi
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  4. Takashi Maoka
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  9. Norihiko Misawa
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Correspondence to Miho Takemura.

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Takemura, M., Kubo, A., Higuchi, Y. et al. Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli. Appl Microbiol Biotechnol 103, 9393–9399 (2019). https://doi.org/10.1007/s00253-019-10182-w

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