Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

Abstract

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches.

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Figure 1: Microbial production of (R,S)-reticuline.
Figure 2: Effects of enzyme levels, substrate levels and culture time on reticuline production levels.
Figure 3: A new strategy for tuning enzyme expression levels.
Figure 4: Microbial production of BIA metabolites along the sanguinarine and berberine branches.
Figure 5: Microbial production of morphinan alkaloids.

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Acknowledgements

We thank P. Facchini (University of Calgary), F.P. Guengerich (Vanderbilt University) and D. Pompon (Centre de Génétique Moléculaire, CNRS) for generously providing cDNAs and yeast strains used in this work. This work was supported by the Center for Biological Circuit Design at Caltech (fellowship to K.M.H.) and the US National Institutes of Health (grant to C.D.S.).

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K.M.H. designed and performed research, analyzed data and wrote the paper; C.D.S. designed research, analyzed data and wrote the paper.

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Correspondence to Christina D Smolke.

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The authors declare competing financial interests in the form of a pending patent application.

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Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Methods (PDF 283 kb)

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Hawkins, K., Smolke, C. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 4, 564–573 (2008). https://doi.org/10.1038/nchembio.105

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