酿酒酵母羊毛甾醇合酶基因单倍体缺陷型突变株的构建

Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene

羊毛甾醇合酶基因(lanosterol synthase gene,erg7)编码的羊毛甾醇合酶是酿酒酵母(Saccharomyces cerevisiae)甾醇生物合成途径中重要的限速酶。酿酒酵母固有的甲羟戊酸(MVA)/麦角甾醇代谢途径的中间体2,3-氧化鲨烯也是三萜类化合物的合成前体。因此,羊毛甾醇合酶催化的2,3-氧化鲨烯环化是麦角甾醇和三萜类化合物生物合成分支形成的关键位点。下调2,3-氧化鲨烯流向麦角甾醇的代谢,可为应用合成生物学技术在酿酒酵母中组建三萜类化合物的代谢途径奠定基础。本研究通过设计含有与erg7基因ORF序列两侧同源的长引物,以质粒PUG66为模板进行PCR,构建带有loxP-Marker-loxP的erg7基因敲除组件,采用LiAc/SS Carrier DNA/PEG方法转化双倍体野生型酿酒酵母INVSc1,通过同源重组的方式构建酿酒酵母erg7基因单倍体缺陷型突变株。半定量PCR和实时定量PCR结果表明,突变株内erg7基因表达水平下降约1倍。TLC和HPLC结果表明,突变株内麦角甾醇含量下降至野生型菌株的42%。

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate （MVA）/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg70RF and plasmid pUG66. The cassette was transformed into diploid wild strain INVScl by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.