Engineering Saccharomyces cerevisiae for high-efficient production of ursolic acid via cofactor engineering and acetyl-CoA optimization

Ursolic acid (UA) is an important plant-derived pentacyclic triterpene with many physiological and pharmaco-logical activities. Although the heterologous biosynthesis of UA in microbes has been achieved, the titer is too low to be applied for commercial industrialization. The low efficiency of key enzymes such as cytochrome P450 enzyme (CYP450) and the imbalance between endogenous metabolism and exogenous pathways are considered key elements. To solve the problem, high-efficient CYP450s and its compatible cytochrome P450 reductases (CPRs) were screened and characterized. Ej8656, a CYP450 from Eriobotrya japonica, combined with LjCPR, a CPR from Lotus japonicus, showed the best performance in an engineered alpha-amyrin producing Saccharomyces cerevisiae with a UA titer of 43.0 mg/L. Furthermore, combining cofactor engineering of NADH/NADPH and optimization of acetyl-CoA supply, UA production was improved to 61.0 mg/L. In addition, fermentation opti-mization was carried out using the constructed S. cerevisiae and the titer of UA was increased to 90.0 mg/L. Finally, 2.33 g/L UA and 1.21 g/L alpha-amyrin were obtained after scale-up experiment in a 5 L fermenter. The UA production was 70.0-fold of the original strain WN1, and is the highest titer reported in S. cerevisiae. Our study provides a combined way to improve the efficiency of heterologous biosynthesis of UA and offers new ideas and methods for the efficient synthesis of other triterpenoids.