Selection and functional identification of berberine biosynthetic genes in <i>Berberis amurensis</i>

Berberine, a pharmacologically important benzylisoquinoline alkaloid (BIA) exhibiting antimicrobial and anti-inflammatory properties, accumulates abundantly in <italic>Berberis amurensis</italic>. However, there is a lack of reports concerning the berberine biosynthesis pathway in <italic>B. amurensis</italic>. In this study, berberine biosynthetic genes were systematically identified in <italic>B. amurensis, </italic>and key functional enzymes (BaNCS4, Ba6OMT4, Ba4'OMT2, and BaSOMT1) were characterized through transcriptomic and biochemical analyses. Notably, Ba4'OMT2 was found to exhibit superior catalytic efficiency compared to its ortholog Cc4'OMT, highlighting its potential as a valuable molecular tool for heterologous biosynthesis platforms. These findings not only elucidate the key enzymes of BIA skeleton formation and berberine <italic>O</italic>-methylation modification, as well as their evolutionary conservation but also offer potential molecular elements for achieving the biosynthesis of berberine through synthetic biology approaches.