Elucidation and Reconstitution of the Andrographolide Biosynthetic Pathway in <i>Saccharomyces cerevisiae</i>

Andrographolide is a bioactive diterpenoid with diverse physiological functions, yet its limited natural supply has hindered further research. In this study, we discovered its natural biosynthetic pathway, which was previously hypothesized to originate from <i>ent</i>-copalol via CYP450-mediated steps. Transcriptomics analyses based on RNA-Seq across different plant tissues of <i>Andrographis paniculata</i> revealed seven CYP450 candidates potentially involved in its biosynthesis from <i>ent</i>-copalol. While individual expression of these enzymes in <i>Saccharomyces cerevisiae</i> did not yield detectable intermediates, coexpression of <i>Ap</i>CYP71A8 and <i>Ap</i>CYP71D10, which exhibited the strongest binding interactions with <i>ent</i>-copalol, achieved conversion of <i>ent</i>-copalol to 3,15,19-trihydroxy-8(17),13-<i>ent</i>-labdadien-16-oic acid. <i>Ap</i>AOP1.2 was found to catalyze the lactone ring formation. Expression of <i>Ap</i>CYP72A219 alone converted 14-deoxyandrographolide to andrographolide. Finally, coexpression of <i>Ap</i>CYP71A8, <i>Ap</i>CYP71D10, <i>Ap</i>AOP1.2, and <i>Ap</i>CYP72A219 achieved <i>de novo</i> biosynthesis of andrographolide in <i>S. cerevisiae</i>. The results provide a strategy for elucidating the biosynthetic pathways of diterpenes and facilitating their heterologous microbial production.