Synthesis of Morphinan Alkaloids in <i>Saccharomyces cerevisiae</i>

<div><p>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 <i>Saccharomyces cerevisiae</i> starting from the precursor (<i>R</i>,<i>S</i>)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (<i>S</i>)-reticuline starting from (<i>R</i>,<i>S</i>)-norlaudanosoline. In addition, the <i>P</i>. <i>somniferum</i> enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in <i>S</i>. <i>cerevisiae</i> and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-<i>O</i>-acetate and synthesis of thebaine from (<i>R</i>)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (<i>R</i>)-reticuline. Yeast cell feeding assays using (<i>R</i>)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-<i>O</i>-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in <i>S</i>. <i>cerevisiae</i> and pave the way for their complete synthesis in recombinant microbes.</p>