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Selection of the optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli

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Abstract

We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via l-DOPA, which is synthesized by hydroxylation of l-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for l-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 μM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes.

The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli

Key points

• There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase.

• Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli.

• New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.

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Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

Professor Taek Soon Lee (Lawrence Berkeley National Laboratory) kindly gifted PCD and DHPR genes. We thank Dr. Yasuharu Satoh (Hokkaido University) for advice and discussion.

Funding

This work was supported by the Project P16009, Development of Production Techniques for Highly Functional Biomaterials Using Smart Cells of Plants and Other Organisms (Smart Cell Project), from the New Energy and Industrial Technology Development Organization (NEDO to H.M.). This work was partly supported by the “Science and Technology that Create New Industries” of the Cannon Foundation and Grant-Support of Asahi Glass Foundation (to A.N.).

Author information

Authors and Affiliations

  1. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-shi, Ishikawa, Japan

    Akira Nakagawa, Eitaro Matsumura, Yurino Yashima, Mizuki Takao & Hiromichi Minami

  2. TechnoPro, Inc., Roppongi Hills Mori Tower 35th floor, 6-10-1 Roppongi, Minatoku, Tokyo, Japan

    Shinya Nakamura

  3. Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

    Sachiyo Aburatani & Katsuro Yaoi

  4. Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

    Takane Katayama

Authors
  1. Akira Nakagawa
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  2. Shinya Nakamura
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  3. Eitaro Matsumura
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  4. Yurino Yashima
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  5. Mizuki Takao
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  6. Sachiyo Aburatani
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  7. Katsuro Yaoi
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  8. Takane Katayama
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  9. Hiromichi Minami
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Contributions

A.N. and H.M. conceived and designed all experiments. A.N., S.N., E.M., Y.Y., and M.T. performed the experiments. S.A. and K.Y. chose the candidate enzymes. A.N, H.M, and T.K. discussed the results. A.N., T.K., and H.M. wrote the manuscript. All the authors reviewed the manuscript.

Corresponding author

Correspondence to Hiromichi Minami.

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Nakagawa, A., Nakamura, S., Matsumura, E. et al. Selection of the optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli. Appl Microbiol Biotechnol 105, 5433–5447 (2021). https://doi.org/10.1007/s00253-021-11401-z

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