Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid “sterol surrogate” tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae. Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.

中文翻译：

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角鲨烯-四氢香豆醇环化酶表达使酿酒酵母能够不依赖于甾醇生长。

固醇的生物合成实际上被认为是所有真核细胞膜的重要组成部分，需要分子氧。因此，酵母酿酒酵母的厌氧生长严格取决于合成生长培养基的甾醇补充。新callimastigomycota是一组严格厌氧的真菌，它们不包含固醇，而是包含由角鲨烯环化形成的五环三萜类“甾醇替代物”四羟木酚。在这里，我们证明了从纤毛虫四膜虫嗜热菌中角鲨烯-四羟木酚环化酶基因TtTHC1的表达使酿酒酵母能够合成四氢香酚。此外，TtTHC1的表达在不含固醇的合成培养基中实现了厌氧啤酒酵母培养物的指数生长。从表达TtTHC1的酿酒酵母菌株中删除ERG1基因后，天然固醇合成被取消，并且在厌氧和有氧条件下均显示了无固醇的持续生长。在不含固醇的培养基上表达TtTHC1的啤酒酵母的厌氧培养物比补充麦角固醇的培养物显示出较低的比生长速率和生物量产量，而其乙醇产率较高。这项研究表明，获得功能性角鲨烯-四氢香豆素环化酶基因可为酿酒酵母提供直接的生长优势。 在固醇受限的厌氧条件下，为消除酵母中与固醇合成相关的氧气需求的代谢工程策略提供了基础。