构建葡萄糖二酸指示系统筛选肌醇加氧酶突变株  

Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants

作　　者：王毳[1] 刘叶[1] 巩旭[1] 刘龙[1,2] 康振 Cui Wangl;Ye Liu;Xu Gong;Long Liu;Zhen Kang(Key Laboratory oflndustrial Biotechnology,Ministry of Education,School ofBiotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School ofBiotechnology,Jiangnan University Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2018年第11期1772-1783,共12页Chinese Journal of Biotechnology

基　　金：轻工技术与工程国家一流学科项目(No.LITE2018-16);长江学者和创新团队发展计划(No.IRT_15R26)资助~~

摘　　要：葡萄糖二酸是一种高附加值天然有机酸,已经广泛应用于疾病防治、生产聚合物材料等领域。在葡萄糖二酸的合成途径中,肌醇加氧酶MIOX所催化的肌醇转换为葡萄糖醛酸的过程是整个途径的限速步骤。通过应用将葡萄糖二酸浓度与绿色荧光蛋白荧光强度相结合的筛选系统,从突变体文库中筛选出3株有潜力的肌醇加氧酶突变体(K59V/R60A、R171S和D276A),使MIOX活性得到提高。重组菌株Escherichia coli BL21(DE3)/MU-R171S的葡萄糖二酸产量相比于未突变菌株提高了36.5%。Glucaric acid（GA）, a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants（K59 V/R60 A, R171 S and D276 A） were screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21（DE3）/MU-R171S accumulated more GA, 136.5% of that of the parent strain.

关 键 词：葡萄糖二酸 肌醇加氧酶 生物传感器 高通量筛选 

分 类 号：TQ925[轻工技术与工程—发酵工程]