α-香树脂醇、羽扇豆醇和计曼尼醇作为重要的五环三萜化合物,具有抗炎、抗肿瘤、抗病毒等多种药理学功能。为构建五环三萜酿酒酵母细胞工厂,该研究运用CRISPR/Cas9技术,分别将编码长春花来源的α-香树脂醇合酶、洋甘草来源的羽扇豆醇合酶和羊蹄甲来源的计曼尼醇合酶的基因整合至酿酒酵母基因组上,获得工程菌株S01、S02和S03。工程菌株在YPG培养基中发酵48 h,S01能生产50.7 mg/L α-香树脂醇,S02羽扇豆醇产量达到45.8 mg/L,S03计曼尼醇产量为17.6 mg/L,表明五环三萜酿酒酵母细胞工厂构建成功。进一步在S01、S02和S03中过表达甲羟戊酸途径中的关键酶:鲨烯环氧酶,增加前体物质2,3-氧化鲨烯的供应,最终α-香树脂醇、羽扇豆醇和计曼尼醇产量分别达96.3、91.6和38.7 mg/L。该研究建立了三萜微生物合成平台,为构建五环三萜酸及其衍生物酿酒酵母细胞工厂奠定了理论和技术基础。
α-Amyrin, lupeol and germanicol are important pentacyclic triterpenoids, with excellent anti-inflammatory, anti-tumor, anti-virus activities and properties. To construct cell factories for the production of pentacyclic triterpenoids, genes encoding Catharanthus roseus α-amyrin synthase (CrαAS), Glycyrrhiza glabra lupeol synthase (GgLUS) and Bauhinia forficata germanicol synthase (BfGES) were separately integrated into the Saccharomyces cerevisiae chromosome using CRISPR/Cas9 technology, resulting in engineered strains S01, S02, and S03. After 48 h of fermentation in YPG medium, 50.7 mg/L α-amyrin, 45.8 mg/L lupeol and 17.6 mg/L germanicol were achieved with strain S01, S02, and S03, respectively. This demonstrated the successful construction of the S. cerevisiae cell factories for pentacyclic triterpenoids production. To increase the supply of precursor 2,3-oxidosqualene, the squalene epoxidase (ERG1), a key enzyme in the mevalonic acid (MVA) pathway, was further overexpressed in strains S01, S02, and S03. With the resulting recombinant strains S04, S05 and S06, the yield of α-amyrin, lupeol and germanicol increased to 96.3, 91.6, and 38.7 mg/L, respectively. Therefore, this study constructed triterpenoid-producing platforms, which provided a theoretical and technical basis for the construction of S.cerevisiae pentacyclic triterpenoid acids cell factories with potential for industrial applications.
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