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

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%。

关键词:葡萄糖二酸，肌醇加氧酶，生物传感器，高通量筛选

Abstract:

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 (K59V/R60A, R171S and D276A) 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.

Key words:glucaric acid, Myo-inositol oxygenase, biosensor, high-throughput screening