Discovery of high-efficiency (S)-N-methylcoclaurine 3′-hydroxylase involved in the biosynthesis of benzylisoquinoline alkaloids in Berberidaceae

Benzylisoquinoline alkaloids (BIAs) represent a class of plant-derived compounds with significant pharmacological importance. The biosynthesis of most BIAs originates from a common precursor (S)-reticuline. (S)-N-methylcoclaurine 3’-hydroxylase (NMCH), encoded by CYP80B members, has always been regarded as a rate-limiting step that restricts the efficient synthesis of (S)-reticuline, thereby leading to the low accumulation of BIAs. The highly-efficiency NMCH still needs to be further explored. Species within the Berberidaceae family are known to accumulate diverse BIAs at relatively high levels, making them an ideal plant resource for exploring highly active enzymes involved in BIA synthesis. Here, by integrating transcriptome and metabolite analysis across 11 Berberidaceae plants, we mine and characterize candidate genes involved in (S)-reticuline biosynthesis. We further establish an engineered yeast platform for functional screening of NMCH genes and evaluate the catalytic activity of all CYP80B candidates. Among these, MbNMCH, isolated from Mahonia bealei, exhibites significantly higher catalytic activity for (S)-reticuline production in yeast compared to previously reported NMCH enzymes. Our findings provide abundant genetic and metabolic information on Berberidaceae plants and identify a highly efficient enzymatic tool for BIA production in microbial cell factories, facilitating the sustainable manufacturing of diverse valuable alkaloids.