Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance

Bioethanol is an environment friendly and renewable source of energy produced by the fermentation of agricultural raw material by a variety of microorganisms including yeast. Obtaining yeast strains that are tolerant to stresses like high levels of ethanol and high temperature is highly desirable as it reduces cost and increases yield during bioethanol production. Here, we report that heterologous expression of C-5 Sterol desaturase (FvC5SD)—an ergosterol biosynthesis enzyme from an edible mushroom Flammulina velutipes in fission yeast, not only imparts increased thermotolerance but also tolerance towards high ethanol concentration and low pH. This tolerance could be attributed to an increase of ≈1.5 fold in the level of ergosterol and oleic acid (C-18 unsaturated fatty acid) as analysed by gas chromatography- mass spectrometry. FvC5SD is a membrane localized iron binding enzyme that introduces double bond at C-5 position into the Δ7-sterol substrates to yield Δ5, 7- sterols as products. In F. velutipes, FvC5SD transcript was observed to be upregulated by ≈5 fold under low pH condition and by ≈ 9 folds and ≈5 fold at 40°C and 4°C respectively when compared to normal growth temperature of 23°C. Besides, susceptibility to cell wall inhibiting drugs like Congo red and Calcoflour white was also found to increase in FvC5SD expressing S. pombe strain. Alteration in membrane sterol and fatty acid composition could also lead to increase in susceptibility to cell wall inhibiting drugs. Thus, this study has immense industrial application and can be employed to ensure competitiveness of fermentation process.

生物乙醇是一种环境友好的可再生能源，可通过包括酵母菌在内的多种微生物发酵农业原料制备得到。获取能够耐受高乙醇浓度、高温等胁迫的酵母菌菌株，可降低生物乙醇生产过程中的成本并提升产物得率，因此具有重要的应用价值。本研究发现，将源自食用真菌金针菇（Flammulina velutipes）的麦角固醇生物合成酶——C-5甾醇去饱和酶（C-5 Sterol desaturase，简称FvC5SD）在裂殖酵母中异源表达后，不仅可增强菌株的耐热性，还能使其耐受高乙醇浓度与低pH环境。经气相色谱-质谱联用（gas chromatography-mass spectrometry）分析，该耐受性的提升可归因于麦角固醇与油酸（C-18不饱和脂肪酸）的含量分别提升约1.5倍。FvC5SD是一种定位于细胞膜的铁结合酶，可在Δ7-甾醇底物的C-5位引入双键，生成Δ5,7-甾醇产物。在金针菇中，与正常生长温度23℃相比，FvC5SD的转录本在低pH条件下上调约5倍，在40℃与4℃条件下分别上调约9倍与5倍。此外，表达FvC5SD的粟酒裂殖酵母（S. pombe）菌株对刚果红（Congo red）、荧光增白剂（Calcofluor white）等细胞壁抑制类药物的敏感性也有所提升。细胞膜甾醇与脂肪酸组成的改变，同样可能导致菌株对细胞壁抑制类药物的敏感性上升。综上，本研究具有极高的工业应用价值，可用于提升发酵工艺的市场竞争力。