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

在这项研究中，构建具有高细胞表面酶活性的酵母菌株，一种新的细胞表面展示系统，基于FLO 1基因编码的凝集素样细胞壁蛋白（Flo 1 p）的酿酒酵母，进行了研究。主要研究内容如下：1.利用酿酒酵母（S.cerevisiae），分别展示葡糖淀粉酶和α-淀粉酶两种淀粉分解酶和内切葡聚糖酶、纤维二糖水解酶和β-葡糖苷酶三种纤维素分解酶，实现了玉米淀粉和纤维素原料直接高效发酵生产乙醇; 2.利用Flo 1 p蛋白，将重组根霉脂肪酶（ProROL）展示于细胞表面，成功催化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能够在非水有机溶剂中高效催化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Lip 3脂肪酶的关系。为探讨GPI锚结构与β-1，6-葡聚糖合成之间的关系，筛选了GPI合成相关基因缺失突变体，以进行渗透补救生长。mcd 4缺失导致GPI细胞壁蛋白水平降低，突变导致细胞壁中甘露聚糖水平降低和碱不溶性β-1，6-葡聚糖和几丁质水平增加。

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.