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.

Fig 1. Description of the (R)-reticuline to morphine biosynthetic pathway reconstituted in S. cerevisiae.

The pathway is divided in two blocks of sequential enzymes all from P. somniferum. The thebaine block includes the enzymes involved in the synthesis of thebaine from (R)-reticuline: PsSAS, salutaridine synthase; PsCPR, cytochrome P450 reductase; PsSAR, salutaridinol reductase; PsSAT, salutaridinol acetyltransferase. The morphine block is composed of enzymes involved in the synthesis of morphine from thebaine: PsT6ODM, thebaine 6-O-demethylase; PsCOR, codeinone reductase; PsCODM, codeine-O-demethylase. Boxed text identifies intermediates used as feeding substrates to test for functional expression of the assembled pathways in yeast.

Fig 2. Reticuline production and utilization in engineered S. cerevisiae.

Biochemical pathway depicting reticuline production and utilization in (a) opium poppy and (b) engineered S. cerevisiae. (c) Immunoblot analysis of recombinant PsSAS (56 kDa) and PsCPR (76 kDa) expression in S. cerevisiae. GAPDH (36 kDa) was used as a loading control. (d) Reticuline production and utilization by strains expressing a recombinant reticuline-producing pathway (strain GCY1086) as well as PsSAS (strain GCY1357), PsBBE (strain GCY1359) or a complete dihydrosanguinarine pathway (strain GCY1125; [4]).

Fig 3. Chiral analysis of reticuline produced from (R,S)-norlaudanosoline by engineered S. cerevisiae.

HPLC-MS chromatographic profile of authentic standards of (a) (R)-reticuline, (b) (S)-reticuline and (c) a mixture of (S)- and (R)-reticuline. (d) Chiral analysis of reticuline produced from (R,S)-norlaudanosoline in cell feeding assays of strain GCY1125 expressing the opium poppy Ps6OMT, PsCNMT, Ps4’OMT and a complete dihydrosanguinarine pathway [4]. (e) Chiral analysis of reticuline produced from (R,S)-norlaudanosoline in cell feeding assays of strain GCY1086 expressing the opium poppy Ps6OMT, PsCNMT and Ps4’OMT. (f) Methylation pathway for conversion of (R,S)-norlaudanosoline to (S)-reticuline.

Fig 4. Synthesis of thebaine in engineered S. cerevisiae.

Synthesis of thebaine from cell feeding assays at pH 7.5, 8, 8.5 or 9 and supplemented with (a) 100 μM (R)-reticuline or (b) 100 μM salutaridine. Strain GCY368 was used in all cell feeding assays. Error bars represent standard deviations of n = 3.

Fig 5. Synthesis of codeine and morphine in S. cerevisiae.

(a) Schematic representation of the production of morphine from thebaine proceeding through the intermediate codeine. Thebaine is demethylated to neopinone by T6ODM. Neopinone spontaneously rearranges to codeinone or is reduced to the side product neopine by COR. Codeine and neopine are demethylated to morphine and the undesired side-product neomorphine by CODM. (b) Synthesis of codeine and morphine from whole cell feeding assays performed at pH 9 and supplemented with 100 μM (R)-reticuline, 100 μM salutaridine or 100 μM codeine. Strain GCY1358 was used in all cell feeding assays. Error bars represent standard deviations of n = 3. * Indicates the substrate used in the cell feeding assays.