De novo biosynthesis of betulinic acid in engineered Saccharomyces cerevisiae

Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae , and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla -derived lupeol C-28 oxidase (BPLO) and Arabidopsis -derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae .