Chromosome-level genomics and cross-species enzyme screening enable high-titer bakuchiol production in Yarrowia lipolytica

Abstract

The diversity of enzymes supporting bakuchiol biosynthesis remains incompletely characterized, limiting the repertoire available for microbial pathway engineering. Here, we combined chromosome-level genomics, cross-species enzyme screening and metabolic engineering to establish high-titer bakuchiol production in Yarrowia lipolytica. We assembled a 471.76-Mb Psoralea corylifolia genome anchored to 11 pseudochromosomes, with a contig N50 of 40.33 Mb. Genome-guided screening and functional validation identified four prenyltransferases from P. corylifolia and Vigna radiata whose N-terminally truncated variants supported bakuchiol production in yeast. These results extend experimental evidence for bakuchiol-producing activity across legume prenyltransferase sequences and species. For PcPT6, corresponding to the previously reported PcPT07, removing 40 N-terminal amino acids increased bakuchiol production by 53% relative to the full-length enzyme. We reconstructed bakuchiol biosynthesis in Y. lipolytica and reinforced aromatic precursor synthesis to increase p-coumaric acid supply. Coordinating mevalonate pathway reinforcement with increased t40PcPT6 gene dosage further improved bakuchiol production. Following fermentation optimization, titers reached 108.29 mg/L in shake flasks and 6.03 g/L in a 7.5-L fed-batch bioreactor. These findings connect an expanded set of functional prenyltransferases with coordinated pathway engineering, enabling gram-per-liter microbial bakuchiol production.