三萜类化合物是一类具有高附加值的次生代谢产物,羊毛甾醇是其生物合成的重要骨架化合物。为了在大肠杆菌中构建羊毛甾醇合成途径,将酵母来源的鲨烯合酶,荚膜甲基球菌来源的鲨烯环氧酶和氧化鲨烯环化酶分别克隆到载体pET28a(+)和pCDF-DUET-1中,共转入大肠杆菌BL21(DE3)底盘细胞,获得了羊毛甾醇生产菌株BT-tSSE-DXOSC。通过过表达甲基赤藓糖醇磷酸(2-C-methyl-D-erythrito-4-phosphate,MEP)途径关键酶1-脱氧-D-木酮糖-5-磷酸合酶和法尼基焦磷酸合酶,增加了前体供应,使羊毛甾醇产量达2.39 mg/L,较优化前提高了2.1倍。通过发酵条件优化,确定了最优pH为7.5,诱导剂IPTG浓度0.5 mmol/L,诱导温度30 ℃,获得羊毛甾醇产量为5.12 mg/L,较初始条件提高了1.1倍。该研究首次构建了产羊毛甾醇的大肠杆菌工程菌株,为以羊毛甾醇为中间体的三萜类物质生物合成及相关代谢途径的研究搭建了平台。
Triterpenoids are a class of bioactive secondary metabolites with high added value, widely used in the pharmaceutical and healthcare industries, and lanosterol is the main skeleton for the biosynthesis of triterpenoids. To construct the lanosterol biosynthetic pathway in Escherichia coli chassis, squalene synthase (SQS) derived from Saccharomyces cerevisiae, squalene epoxidase (SE) and oxidosqualene cyclase (OSC) derived from Methylococcus capsulatus were cloned into vectors pET28a (+) and pCDF-DUET-1 respectively, and co-transformed into E. coli BL21 (DE3). The engineered strain BT-tSSE-DXOSC produced 0.77 mg/L lanosterol. Precursor supply was improved by overexpressing key enzymes in the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and farnesyl pyrophosphate synthase (FPS), thus lanosterol production reached 2.39 mg/L, increasing by 2.1 times compared to that before optimization. The optimal fermentation conditions were determined by single-factor and orthogonal experiments: pH 7.5, isopropyl-b-D-thiogalactoside (IPTG) concentration 0.5 mmol/L, and inducing temperature 30 ℃, under which the lanosterol production reached 5.12 mg/L, increasing by 1.1 times than that under initial conditions. This study constructed a lanosterol-producing strain for the first time, which provides a platform for the synthesis of triterpenoids and steroids as well as the study of relating metabolic pathways.
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