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

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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Figure 1: Development and characterization of an enzyme-coupled L-DOPA biosensor.
Figure 2: Isolation and improvement of a tyrosine hydroxylase in yeast.
Figure 3: Characterization of reduced DOPA oxidase activity in CYP76AD1 mutants.
Figure 4: Production of (S)-reticuline from glucose.

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References

  1. Leonard, E., Runguphan, W., O'Connor, S. & Prather, K.J. Opportunities in metabolic engineering to facilitate scalable alkaloid production. Nat. Chem. Biol. 5, 292–300 (2009).

    CAS  Article  Google Scholar 

  2. Koehn, F.E. & Carter, G.T. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 4, 206–220 (2005).

    CAS  Article  Google Scholar 

  3. Glenn, W.S., Runguphan, W. & O'Connor, S.E. Recent progress in the metabolic engineering of alkaloids in plant systems. Curr. Opin. Biotechnol. 24, 354–365 (2013).

    CAS  Article  Google Scholar 

  4. Paterson, I. & Anderson, E.A. Chemistry. The renaissance of natural products as drug candidates. Science 310, 451–453 (2005).

    Article  Google Scholar 

  5. Mora-Pale, M., Sanchez-Rodriguez, S.P., Linhardt, R.J., Dordick, J.S. & Koffas, M.A.G. Biochemical strategies for enhancing the in vivo production of natural products with pharmaceutical potential. Curr. Opin. Biotechnol. 25, 86–94 (2014).

    CAS  Article  Google Scholar 

  6. Facchini, P.J. et al. Synthetic biosystems for the production of high-value plant metabolites. Trends Biotechnol. 30, 127–131 (2012).

    CAS  Article  Google Scholar 

  7. Arkin, A.P. & Fletcher, D.A. Fast, cheap and somewhat in control. Genome Biol. 7, 114 (2006).

    Article  Google Scholar 

  8. Siddiqui, M.S., Thodey, K., Trenchard, I. & Smolke, C.D. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res. 12, 144–170 (2012).

    CAS  Article  Google Scholar 

  9. Paddon, C.J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013).

    CAS  Article  Google Scholar 

  10. Hagel, J.M. & Facchini, P.J. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672 (2013).

    CAS  Article  Google Scholar 

  11. Beaudoin, G.A.W. & Facchini, P.J. Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240, 19–32 (2014).

    CAS  Article  Google Scholar 

  12. Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. 2, 326 (2011).

    Article  Google Scholar 

  13. Nakagawa, A. et al. (R,S)-Tetrahydropapaveroline production by stepwise fermentation using engineered Escherichia coli. Sci. Rep. 4, 6695 (2014).

    CAS  Article  Google Scholar 

  14. Hawkins, K.M. & Smolke, C.D. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564–573 (2008).

    CAS  Article  Google Scholar 

  15. Fossati, E. et al. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 5, 3283 (2014).

    Article  Google Scholar 

  16. Thodey, K., Galanie, S. & Smolke, C.D. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat. Chem. Biol. 10, 837–844 (2014).

    CAS  Article  Google Scholar 

  17. Minami, H. et al. Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. USA 105, 7393–7398 (2008).

    CAS  Article  Google Scholar 

  18. Mee, M.T., Collins, J.J., Church, G.M. & Wang, H.H. Syntrophic exchange in synthetic microbial communities. Proc. Natl. Acad. Sci. USA 111, E2149–E2156 (2014).

    CAS  Article  Google Scholar 

  19. Zhou, K., Qiao, K., Edgar, S. & Stephanopoulos, G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat. Biotechnol. 33, 377–383 (2015).

    CAS  Article  Google Scholar 

  20. Lichman, B.R. et al. 'Dopamine-first' mechanism enables rational engineering of norcoclaurine synthase aldehyde activity profile. FEBS J. 282, 1137–1151 (2015).

    CAS  Article  Google Scholar 

  21. Fitzpatrick, P.F. Tetrahydropterin-dependent amino acid hydroxylases. Annu. Rev. Biochem. 68, 355–381 (1999).

    CAS  Article  Google Scholar 

  22. Claus, H. & Decker, H. Bacterial tyrosinases. Syst. Appl. Microbiol. 29, 3–14 (2006).

    CAS  Article  Google Scholar 

  23. Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M. & Lomascolo, A. Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. J. Appl. Microbiol. 100, 219–232 (2006).

    CAS  Article  Google Scholar 

  24. Gandía-Herrero, F., García-Carmona, F. & Escribano, J. Botany: floral fluorescence effect. Nature 437, 334 (2005).

    Article  Google Scholar 

  25. Sasaki, N. et al. Detection of DOPA 4,5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa. Plant Cell Physiol. 50, 1012–1016 (2009).

    CAS  Article  Google Scholar 

  26. Gandía-Herrero, F. & García-Carmona, F. Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci. 18, 334–343 (2013).

    Article  Google Scholar 

  27. Gandía-Herrero, F., García-Carmona, F. & Escribano, J. A novel method using high-performance liquid chromatography with fluorescence detection for the determination of betaxanthins. J. Chromatogr. A 1078, 83–89 (2005).

    Article  Google Scholar 

  28. Zhang, J.H., Chung, T. & Oldenburg, K. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  Article  Google Scholar 

  29. Santos, C.N.S. & Stephanopoulos, G. Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli. Appl. Environ. Microbiol. 74, 1190–1197 (2008).

    CAS  Article  Google Scholar 

  30. Lezzi, C., Bleve, G., Spagnolo, S., Perrotta, C. & Grieco, F. Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells. J. Ind. Microbiol. Biotechnol. 39, 1875–1880 (2012).

    CAS  Article  Google Scholar 

  31. Hernández-Romero, D., Sanchez-Amat, A. & Solano, F. A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio. FEBS J. 273, 257–270 (2006).

    Article  Google Scholar 

  32. Hatlestad, G.J. et al. The beet R locus encodes a new cytochrome P450 required for red betalain production. Nat. Genet. 44, 816–820 (2012).

    CAS  Article  Google Scholar 

  33. Gandía-Herrero, F. & García-Carmona, F. Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains. Planta 236, 91–100 (2012).

    Article  Google Scholar 

  34. Koyanagi, T. et al. Eukaryotic-type aromatic amino acid decarboxylase from the root colonizer Pseudomonas putida is highly specific for 3,4-dihydroxyphenyl-L-alanine, an allelochemical in the rhizosphere. Microbiology 158, 2965–2974 (2012).

    CAS  Article  Google Scholar 

  35. Luttik, M.A.H. et al. Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact. Metab. Eng. 10, 141–153 (2008).

    CAS  Article  Google Scholar 

  36. Gandía-Herrero, F., Escribano, J. & García-Carmona, F. Characterization of the activity of tyrosinase on betanidin. J. Agric. Food Chem. 55, 1546–1551 (2007).

    Article  Google Scholar 

  37. Dohm, J.C. et al. The genome of the recently domesticated crop plant sugar beet (Beta vulgaris). Nature 505, 546–549 (2014).

    CAS  Article  Google Scholar 

  38. Pieper, U. et al. ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 42, D336–D346 (2014).

    CAS  Article  Google Scholar 

  39. Sentheshanmuganathan, S. & Elsden, S.R. The mechanism of the formation of tyrosol by Saccharomyces cerevisiae. Biochem. J. 69, 210–218 (1958).

    CAS  Article  Google Scholar 

  40. Pauli, H.H. & Kutchan, T.M. Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3′-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450–dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 13, 793–801 (1998).

    CAS  Article  Google Scholar 

  41. Xiao, M. et al. Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest. J. Biotechnol. 166, 122–134 (2013).

    CAS  Article  Google Scholar 

  42. Minami, H., Dubouzet, E., Iwasa, K. & Sato, F. Functional analysis of norcoclaurine synthase in Coptis japonica. J. Biol. Chem. 282, 6274–6282 (2007).

    CAS  Article  Google Scholar 

  43. Hazelwood, L.A., Daran, J.-M., van Maris, A.J.A., Pronk, J.T. & Dickinson, J.R. The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 74, 2259–2266 (2008).

    CAS  Article  Google Scholar 

  44. Fossati, E., Narcross, L., Ekins, A., Falgueyret, J.P. & Martin, V.J.J. Synthesis of morphinan alkaloids in Saccharomyces cerevisiae. PLoS ONE 10, e0124459 (2015).

    Article  Google Scholar 

  45. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6, e16765 (2011).

    CAS  Article  Google Scholar 

  46. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4, e5553 (2009).

    Article  Google Scholar 

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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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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.

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Correspondence to John E Dueber.

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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.

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DeLoache, W., Russ, Z., Narcross, L. et al. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat Chem Biol 11, 465–471 (2015). https://doi.org/10.1038/nchembio.1816

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