Strong Feedback Inhibition of Key Enzymes in the Morphine Biosynthetic Pathway from Opium Poppy Detectable in Engineered Yeast

Abstract

Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3′-hydroxy-N-methylcoclaurine 4′-methyltransferase (4′OMT), which yields (S)-reticuline, and the coupled salutaridinol-7-O-acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert (S)-norcoclaurine to (S)-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4′OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4′OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.

Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast

“…This slightly promoted sanguinarine synthesis, and as a result, SAN229 exhibited a sanguinarine titer of 97.94 mg L −1 . It has been reported that a class of BIA uptake permeases, known as BIA importers (BUP), localized in the Opium poppy plasma membrane, was able to transport various BIAs and certain pathway precursors (e.g., dopamine) and enhance the uptake rate of codeine and intermediates in yeast cells 54 , 55 . Unfortunately, the introduction of BUP1 into the SAN229 strain failed to significantly increase sanguinarine production, possibly due to mislocalization of BUP1 in the yeast organelle (Supplementary Fig.…”

“…While this study has addressed several bottlenecks in sanguinarine biosynthesis, many aspects of the complex plant secondary metabolite pathway still require further investigation 14 . A recent study reported negative feedback inhibition of Ps 4’OMT by reticuline, which should be addressed by protein engineering efforts to further enhance the metabolic fluxes towards sanguinarine biosynthesis 55 . The establishment of a fluorescence-based high-throughput screening platform will facilitate a more comprehensive and systematic exploration of sanguinarine biosynthesis in yeast (Supplementary Fig.…”