Summary Strigol is the first identified and one of the most important strigolactones (SLs), but the biosynthetic pathway remains elusive. We functionally identified a strigol synthase (cytochrome P450 711A enzyme) in the Prunus genus through rapid gene screening in a set of SL‐producing microbial consortia, and confirmed its unique catalytic activity (catalyzing multistep oxidation) through substrate feeding experiments and mutant analysis. We also reconstructed the biosynthetic pathway of strigol in Nicotiana benthamiana and reported the total biosynthesis of strigol in the Escherichia coli‐yeast consortium, from the simple sugar xylose, which paves the way for large‐scale production of strigol. As proof of concept, strigol and orobanchol were detected in Prunus persica root extrudes. This demonstrated a successful prediction of metabolites produced in plants through gene function identification, highlighting the importance of deciphering the sequence–function correlation of plant biosynthetic enzymes to more accurately predicate plant metabolites without metabolic analysis. This finding revealed the evolutionary and functional diversity of CYP711A (MAX1) in SL biosynthesis, which can synthesize different stereo‐configurations of SLs (strigol‐ or orobanchol‐type). This work again emphasizes the importance of microbial bioproduction platform as an efficient and handy tool to functionally identify plant metabolism.

Strigol was the first strigolactone (SL) to be discovered, but the biosynthetic pathway remains elusive. Here, through rapid gene screening using a microbial SL-producing platform, we functionally identified a strigol synthase (PpMAX1c, a cytochrome P450 711A enzyme) in Prunus that synthesizes strigol directly from the SL precursor carlactone through catalyzing multi-step oxidations and C-ring cyclization, bypassing the synthesis of 5-deoxystrigol. The function of PpMAX1c was validated through reconstructing the biosynthesis of strigol in Nicotiana benthamiana. Additional genomic analysis and functional verification confirm that peach also encodes an orobanchol synthase (PpCYP722C, a cytochrome P450 722C enzyme), which hints at the presence of both strigol-type and orobanchol-type SLs in peach and was confirmed through metabolic analysis of peach seedlings. This work highlights the catalytic diversity of the largely unexplored family of CYP711A homologs and sets the foundation to characterize the roles of different types of SLs in the economically important Prunus.