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，本研究基于编码凝集素样细胞壁蛋白（Flo1p）的FLO1基因，研究了一种新的细胞表面展示系统。得到的几个主题如下：1。短句来源以玉米淀粉和纤维素原料为原料，利用酵母（S.cerevisiae）结合两种淀粉酶（葡萄糖淀粉酶和α-淀粉酶）和三种纤维素水解酶（内切葡聚糖酶、纤维素生物水解酶和β-葡萄糖酶）直接高效地发酵生产乙醇。利用该体系以Flo1p蛋白为载体，将重组米根霉（Rhizopus oryzae, proorol）的重组脂肪酶序列展示在细胞表面，并成功催化了甲醇分解反应，用于生产生物柴油燃料。此外，显示在酵母细胞表面的proorol能在非水有机溶剂中高效催化对映选择性酯交换反应。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。进一步研究了LST-03与Lip3脂肪酶之间的关系。从耐溶剂有机微生物P.aeruginosa中获得PST-01蛋白酶，阐明了PST-01蛋白酶催化反应的动力学和机理。为了探索GPI锚定结构与β-1,6-葡聚糖合成之间的关系，筛选了GPI合成相关基因的缺失突变体，用于渗透补救生长。mcd4缺失导致GPI细胞壁蛋白水平降低，突变导致细胞壁甘露聚糖水平降低，碱不溶性β-1,6-葡聚糖和几丁质水平升高。

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.