Abstract:

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.

Key words: position effect, ursolic acid, Saccharomyces cerevisiae, metabolism, fermentation, synthetic biology

摘要：

熊果酸（UA）是具有多种生理及药理活性的植物来源五环三萜类羧酸，尽管在酿酒酵母中已经实现了UA的从头合成，但仍存在产量低、副产物积累、营养供给冗余等问题，无法实现工业化生产。为了提高酿酒酵母合成熊果酸的能力，通过多种方法优化了UA合成途径中异源CYP450酶的表达。探究了位置效应对CYP450基因表达的影响，初步筛选到CYP450的基因组最佳整合位点（HO位点），通过单独增加CYP450的拷贝数或同时增加CYP450及CPR的拷贝数，确定了同时存在3个CYP450和CPR拷贝时CYP450酶具有最佳催化活性，进行培养基优化后UA摇瓶产量达到（266.54±6.50） mg/L，最终在5 L发酵罐进行放大培养UA产量达到（2825.58±36.26）mg/L。本研究可为在酿酒酵母中通过表达优化策略调控其他三萜类化合物的合成提供参考。

关键词: 位置效应, 熊果酸, 酿酒酵母, 代谢, 发酵, 合成生物学