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

Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3 '-hydroxy-N-methylco-claurine 4 '-methyltransferase (4 ' OMT), which yields (S)-reticu-line, 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.

Automated summary

Systematic screening of morphine pathway intermediates in engineered yeast identified key biosynthetic enzymes that exhibit potent feedback inhibition. Specifically, 4'-O-methyltransferase (4' OMT) produces (S)-reticuline and salutaridinol-7-O-acetyltransferase (SalAT) and thebaine synthase (THS2) form the enzyme system responsible for the synthesis of thebaine. Addition of deuterated reticuline-d1 to a yeast strain capable of converting (S)-norcoclaurine to (S)-reticuline resulted in reduced product accumulation. Similar effects were observed with deuterated thebaine-d3 and a yeast strain able to convert salutaridine to thebaine. In vitro analysis showed that reticuline inhibits 4' OMT in a noncompetitive manner, while thebaine exerts mixed inhibition on SalAT/THS2. Furthermore, the addition of reticuline and thebaine led to the accumulation of pathway intermediates in a yeast strain engineered for de novo morphine biosynthesis, suggesting that blocking the interaction of these compounds with their respective enzymes could significantly increase opiate alkaloid production in engineered yeast.