title: Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.
abstract: Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.
authors.affiliation: Department of Biology, Concordia University, Montréal, Québec, Canada; Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Elena
authors.initials: E
authors.lastname: Fossati
authors.affiliation: Department of Biology, Concordia University, Montréal, Québec, Canada; Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Lauren
authors.initials: L
authors.lastname: Narcross
authors.affiliation: Department of Biology, Concordia University, Montréal, Québec, Canada; Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Ekins
authors.affiliation: Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
authors.affiliation: Department of Biology, Concordia University, Montréal, Québec, Canada; Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Vincent J J
authors.initials: VJ
authors.lastname: Martin
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005583:Fourier Analysis; D013058:Mass Spectrometry; D009019:Morphinans / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D009019:Morphinans