异源 CYP450 酶的表达优化促进工程酿酒酵母合成熊果酸

Ursolic acid (UA) is a plant pentacyclic triterpenoid carboxylic acid with a variety of physiological and pharmacological activities. Although the de novo synthesis of UA has been achieved in Saccharomyces cerevisiae, there are still problems such as low yield, by-product accumulation, and redundant nutrient supply, which cannot meet the demand of industrial production. In order to improve the ability of synthesizing ursolic acid in Saccharomyces cerevisiae, the expression of heterologous CYP450 enzymes in the UA synthesis pathway was optimized by different methods. Position effect has an important impact on the expression level of exogenous genes, and the expression ratio of CYP450 and CPR can affect electron transport and thus regulate the catalytic activity of CYP450 enzymes. Therefore, the effect of position effect on CYP450 gene expression was investigated in this study. The optimal integration site (HO site) of CYP450 in the genome was preliminarily selected. By increasing the copy number of CYP450 alone or CYP450 and CPR simultaneously, it was determined that CYP450 enzyme had the best catalytic activity when there were three copies of CYP450 and CPR. After medium optimization, the UA yield in shake flask experiment reached (266.54±6.50) mg/L. Finally, the UA production was scaled up in a 5 L fermentation tank and reached (2825.58±36.26) mg/L. This study provides an reference for regulating the synthesis of other triterpenoids through expression optimization in Saccharomyces cerevisiae.