【摘要】 柠檬烯及其衍生物紫苏酸作为重要的生物活性天然产物，广泛应用于食品、化妆品、保健品和医药等行业。然而，低效率的植物提取与高能耗的化工合成限制了柠檬烯和紫苏酸的工业合成。本研究在酿酒酵母中通过过氧化物酶体区室化表达绿薄荷来源的柠檬烯合酶，构建获得重组菌株，柠檬烯产量为0.038 mg/L。采用模块化工程分步表达参与柠檬烯合成的基因ERG10、ERG13、tHMGR、ERG12、ERG8、IDI1、MVD1、ERG20ww以及tLS，以研究其对柠檬烯产量的影响。通过增加前体模块，柠檬烯产量增加至1.14 mg/L。采用高拷贝数的质粒表达上述关键基因，柠檬烯的产量显著提高，达到86.74 mg/L，提高至初始菌株产量的4 337倍。以构建的柠檬烯生产菌株为出发菌株，通过表达丹参来源的细胞色素P450酶基因，实现了紫苏酸的生成，其产量达4.42 mg/L，为利用酿酒酵母构建高产单萜类天然产物的细胞工厂奠定了基础。更多还原

【Abstract】 Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However,inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13,tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L,which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.更多还原