Table_1_Crocetin Overproduction in Engineered Saccharomyces cerevisiae via Tuning Key Enzymes Coupled With Precursor Engineering.DOCX

Crocetin, an important natural carotenoid dicarboxylic acid with high pharmaceutical values, has been successfully generated from glucose by engineered Saccharomyces cerevisiae in our previous study. Here, a systematic optimization was executed for crocetin overproduction in yeast. The effects of precursor enhancement on crocetin production were investigated by blocking the genes involved in glyoxylate cycle [citric acid synthase (CIT2) and malic acid synthase (MLS1)]. Crocetin titer was promoted by 50% by ΔCIT2 compared to that of the starting strain. Then, the crocetin production was further increased by 44% through introducing the forward fusion enzymes of PsCrtZ (CrtZ from Pantoea stewartii)-CsCCD2 (CCD2 from Crocus sativus). Consequently, the crocetin titer reached to 1.95 ± 0.23 mg/L by overexpression of PsCrtZ-CsCCD2 followed by medium optimization. Eventually, a titer of 12.43 ± 0.62 mg/L crocetin was achieved in 5-L bioreactor, which is the highest crocetin titer reported in micro-organisms.

番红花酸（Crocetin）是一类兼具高药用价值的重要天然类胡萝卜素二羧酸，本团队前期研究已通过工程化酿酒酵母（Saccharomyces cerevisiae）实现了以葡萄糖为底物的番红花酸合成。本研究针对酿酒酵母中番红花酸的过量合成开展系统性优化：首先通过敲除乙醛酸循环相关基因——柠檬酸合酶（CIT2）与苹果酸合酶（MLS1），探究了前体供给增强对番红花酸合成的影响。结果显示，CIT2基因敲除（ΔCIT2）可使番红花酸效价较出发菌株提升50%；随后引入斯氏泛菌（Pantoea stewartii）来源的CrtZ与番红花（Crocus sativus）来源的CCD2的正向融合酶，进一步将番红花酸产量提升44%。在此基础上，通过过表达该融合酶并结合培养基优化，菌株的番红花酸效价达到1.95 ± 0.23 mg/L；最终在5-L生物反应器中，番红花酸效价可达12.43 ± 0.62 mg/L，为目前已报道的微生物源番红花酸最高效价。