Research of construction of bi-functional reaction field on cell-surface and its application

为了构建具有高细胞表面酶活性的酵母菌株，基于酿酒酵母中编码凝集素样细胞壁蛋白(Flo1p)的FLO1基因构建了一种新型的细胞表面展示系统。主要研究内容如下：1.以玉米淀粉和纤维素原料为原料，利用具有两种淀粉酶和α-淀粉酶以及三种纤维素酶的酿酒酵母，实现了直接高效发酵生产乙醇。2.利用该系统，利用Flo1p蛋白，在细胞表面展示了米根霉脂肪酶(ProROL)，并成功地催化了甲醇分解反应生产生物柴油。此外，展示在酵母细胞表面的ProROL能有效地催化非水有机溶剂中的对映选择性酯交换反应。3.从耐有机溶剂的铜绿假单胞菌LST-03中克隆了一个脂肪酶基因(Lip 3)，并在大肠杆菌中表达。此外，还探讨了LST-03与Lip3脂肪酶之间的关系。研究了耐有机溶剂型铜绿假单胞菌Pst-01酶催化β-1，6-葡聚糖合成的动力学和机理。4.筛选了参与渗透补救性生长的葡聚糖合成相关基因的缺失突变体。MCD4缺失导致细胞壁蛋白水平下降，突变导致细胞壁中甘露聚糖水平下降，碱不溶β-1，6-葡聚糖和甲壳素水平上升。

In this study, to construct yeast strains with high cell surface enzyme activity, a novel cell-surface display system, based on the FLO1 gene encoding a lectin-like cell-wall protein (Flo1p) in Saccharomyces cerevisiae, was investigated. Several topics obtained are as follows :1.Direct and efficient productions of ethanol by fermentation from raw corn starch and cellulosic material were achieved by using the yeast S.cerevisiae condisplaying two amylolytic enzymes such as glucoamylase and α-amylase and three cellulolytic enzymes such as endoglucanase, cellobiohydrolase and β-glucosodase, respectively.2.Using this system using Flo1p protein, recombinant lipase with a pro sequence from Rhizopus oryzae (ProROL) was displayed on the cell surface, and sucuessfully catalyzed the methanolysis reaction for biodiesel fuel production. Besides, ProROL displayed on the yeast cell surface could efficiently catalyze enantioselective transesterification in non-aqueous organic solvent.3.A lipase gene (lip 3) was cloned from the Pseudomonas aeroginosa strain LST-03, which tolerates organic solvents and expressed in Eschrichi coli. Furtheremore, the relationship between LST-03 and Lip3 lipases was investigated. The kinetics and mechanism of the reaction catalyzed by the P.aeruginosa PST-01 protease, which was obtained from an organic solvent-tolerant microorganism P.aeruginosa, was clarified.4.To explore the relationship between the GPI anchor structure and β-1,6-glucan synthesis, deletion mutants in genes involved in GPI synthesis for osmotic remedial growth were screened. The mcd4 deletion causes a decrease in GPI cell wall proteins levels, and the mutation caused a decrease in mannan levels and increase in alkali-insoluble β-1,6-glucan and chitin levels in the cell wall.