Glycerol improves heterologous biosynthesis of betulinic acid in engineered Yarrowia lipolytica

Glycerol is the main byproduct of the biodiesel production process and can be converted into valuable products by Yarrowia lipolytica. Betulinic acid is a pentacyclic triterpene with a variety of pharmacological properties. Herein we report heterologous biosynthesis of betulinic acid in Y. lipolytica by co-expressing lupeol synthase opAtLUP1, NADPH-cytochrome P450 monooxygenase opCYP716A12, and NADPH-cytochrome P450 reductase opAtCPR1. Initially, the engineered Y. lipolytica produced 0.32 mg/L betulinic acid. This titer was increased to 9.41 mg/L following P450 enzyme fusion and overexpression of key genes from the upstream mevalonate (MVA) pathway. Substitution of glycerol for glucose further enhanced betulinic acid production to 16.98 mg/L, 1.8-fold higher than the titer obtained with glucose. 26.53 mg/L betulinic acid was obtained with 40 g/L glycerol as the sole carbon source, which was comparable to the titer obtained from Saccharomyces cerevisiae with 50 g/L glucose in shake flask cultivation. Glycerol improved betulinic acid titer by increasing the expression of key genes in the MVA pathway, and by increasing the supply of acetyl-CoA. Our study highlights the possible applications of glycerol as carbon source in the production of valuable terpenoids by engineered Y. lipolytica.