甾醇C-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶的编码序列及其终止子序列，以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体，以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG2；同时，在本实验室已构建的ERG6表达质粒pPERG6的基础上，构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58，依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中，甾醇中间体和终产物麦角甾醇的含量均比对照菌高；而在ERG2高表达的酵母菌株中，无论甾醇中间体，还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍，是ERG2单独高表达菌株YS58(pPERG2)的1.92倍，是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤，该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应，而且使麦角甾醇含量进一步提高，为构建麦角甾醇高产酵母工程菌株提供了实验依据。

Regulation role of sterol C-24 methyltransferase and sterol C-8 isomerase in the ergosterol biosynthesis of Saccharomyces cerevisiae

Abstract: Objective] Ergosterol is a fungal metabolite with economic importance. For ergosterol biosynthesis, to identify the bottleneck enzymes in the metabolic pathway is of crucial importance. Methods] Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR. To evaluate the effect of ERG2 overexpression on the sterol content in the yeast, the expression plasmid pPERG2 was constructed and transformed into S. cerevisiae strain YS58 to generate recombinant strain YS58(pPERG2). In addition, the regulation role of sterol C-24 methyltransferase, encoded by ERG6, in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6, ERG2 respectively, or overexpressing ERG6 and ERG2 simultaneously. Results] Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S.cerevisiae. Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis, overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level, which was 1.41-fold of that in empty vector strain, 1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain. Conclusion] These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S. cerevisiae.