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An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose

Nature Chemical Biology volume 11, pages 465471 (2015) | Download Citation

Abstract

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.

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NCBI Reference Sequence

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Acknowledgements

We thank H. Lee for assistance with preliminary experiments; S. Bauer for LC/MS training; members of the Martin and Dueber Labs, in particular M. Lee, for valuable feedback throughout the project and in the preparation of the manuscript; and L. Bourgeois and J. Scrivens for their contribution in identifying NCS enzymes active in yeast. The work on engineering an enzyme-coupled biosensor was supported by the US Department of Energy Office of Science Early Career Research Program (Office of Biological and Environmental Research) under award number DE-SC0008084 (grant to J.E.D.), the US National Science Foundation (fellowship to W.C.D.) and the US Department of Defense (fellowship to Z.N.R.). Research in the Martin lab was financially supported by Genome Canada, Genome Québec and a Canada Research Chair (V.J.J.M.)

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Affiliations

  1. Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.

    • William C DeLoache
    • , Zachary N Russ
    • , Andrew M Gonzales
    •  & John E Dueber
  2. Department of Biology, Concordia University, Montréal, Québec, Canada.

    • Lauren Narcross
    •  & Vincent J J Martin
  3. Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.

    • Lauren Narcross
    •  & Vincent J J Martin

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Contributions

W.C.D., Z.N.R., L.N., V.J.J.M. and J.E.D. designed the research. W.C.D. and Z.N.R. performed the experiments, and L.N. conducted chiral analysis. A.M.G. assisted in preliminary studies. W.C.D., Z.N.R. and L.N. analyzed the results. V.J.J.M. and J.E.D. supervised the research. W.C.D., V.J.J.M. and J.E.D. wrote the manuscript with editing help from Z.N.R. and L.N.

Competing interests

W.C.D., Z.N.R., J.E.D., L.N. and V.J.J.M. declare competing financial interests in the form of a pending patent application, US application no. 62/094,877.

Corresponding author

Correspondence to John E Dueber.

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https://doi.org/10.1038/nchembio.1816

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