利用Flo1p锚定蛋白在酿酒酵母表面展示三种纤维素酶的纤维素乙醇发酵

Display cellulolytic enzymes on Saccharomyces cerevisiae cell surface by using Flo1p as an anchor protein for cellulosic ethanol production

为了简化纤维素乙醇生产工艺,实现纤维素利用与乙醇发酵的同步进行,通过酵母细胞表面展示技术,以酿酒酵母菌株Saccharomyces cerevisiae Y5为受体,通过絮凝素(Flo1p)锚定方式,将来自丝状真菌里氏木霉Trichoderma reesei的内切葡聚糖酶Ⅱ(EGII)、纤维二糖水解酶Ⅱ(CBHII)以及来自棘孢曲霉Aspergillus aculeatus的β-葡糖苷酶Ⅰ(BGLI)展示在细胞表面,构建同时表达3种纤维素酶的酵母菌群系统。经过免疫荧光验证展示酶的细胞蛋白定位,酶活测定,乙醇发酵性能验证,结果表明:展示表达的3种纤维素酶具有良好的稳定性和功能活性;在EGII、CBHII和BGLI协同作用下重组酵母菌株能够水解溶胀磷酸纤维素(Phosphoric acid swollen cellulose,简称PASC)并产生乙醇,乙醇浓度达到最大值0.77 g/L,乙醇产量为0.35 g/g,相当于理论值的68.6%。本研究成功构建了利用Flo1p作为锚定蛋白的絮凝素展示系统,初步实现了纤维素利用与乙醇发酵的同步进行,为利用酿酒酵母表面展示技术固定并表达纤维素酶提供了一定的理论依据。

In this study, we constructed a yeast consortium surface-display expression system by using Flol as an anchor protein. Endoglucanase Ⅱ （EGⅡ） and cellobiohydrolaseⅡ （CBHⅡ） from Trichoderma reesei, and β-glucosidase 1 （BGLI） from Aspergillus aculeatus were immobilized on Saccharomyces cerevisiae Y5. We constructed the cellulose-displaying expression yeast consortium （Y5/fEGII：Y5/fCBHII：Y5/fBGLI=1：1：1） and investigated the enzymatic ability and ethanol fermentation. The displayed cellulolytic enzymes was stabile during the 96-h fermentation. The yeast consortium produced 0.77 g/L ethanol from 10 g/L phosphoric acid swollen cellulose （PASC） within 96 h. The yield （in grams of ethanol produced per gram of carbohydrate consumed） was 0.35 g/g, which correspond to 68.6% of the theoretical yield.