定向进化肌醇加氧酶MIOX4提高葡萄糖二酸的产量  

Directed evolution of inositol oxygenase MIOX4 to increase the glucaric acid production

作　　者：苏蕊芳 李诗韵 毛银 赵运英 邓禹[1,2,3] SU Ruifang;LI Shiyun;MAO Yin;ZHAO Yunying;DENG Yu(National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;Jiangsu Provincial Research Center for Bioactive Product Processing Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学,江苏省生物活性产品加工工程研究中心,江苏无锡214122

出　　处：《食品与发酵工业》2022年第22期1-8,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2019YFA0905502);国家自然科学基金(21877053)。

摘　　要：葡萄糖二酸是一种在医药、食品、服装纺织和化工均有重要作用的有机二元酸。葡萄糖二酸的生物合成途径已经在大肠杆菌、毕赤酵母和酿酒酵母细胞中成功构建。为了提高葡萄糖二酸合成关键酶-肌醇加氧酶MIOX4的活性,该研究以酿酒酵母(Saccharomyces cerevisiae)作为底盘微生物,通过定向进化-易错PCR的方法对来自拟南芥基因组的肌醇加氧酶MIOX4的关键氨基酸序列进行随机突变。以葡萄糖二酸的产量为筛选指标,利用大肠杆菌葡萄糖二酸传感器对MIOX4的突变体进行高通量筛选。实验结果显示,从突变体文库中筛到4株有潜力的MIOX4突变体(S133R、K88R、E72V、T40P),使得葡萄糖二酸产量相较于未突变菌株分别提高了53%、30%、21%、17%。通过对突变体MIOX4(S133R)进行结构分析,发现突变位点R133增大了该位点与底物肌醇之间的空间距离,从而减少了空间位阻,提高了对底物肌醇的催化能力。该研究为后续研究MIOX的结构和功能、以及进一步提高葡萄糖二酸产量奠定了基础。Glucaric acid is an organic dibasic acid that plays an important role in medicine, food, clothing, textile and chemical industry. It is known as one of the top value-added chemicals from biomass. The synthesis of glucaric acid mainly relies on chemical oxidation and biological methods, but the production of glucaric acid by chemical oxidation has disadvantages such as low yield, many by-products, and unfriendly environment problems. The biosynthesis of glucaric acid is more environmentally friendly, and can realize low-cost production of glucaric acid. The biosynthetic pathway of glucaric acid had been successfully constructed in Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae. At present, the production of glucaric acid has been greatly improved by metabolic engineering and synthetic biology methods, but the activity and stability of the key enzyme in the synthesis pathway of glucaric acid, inositol oxygenase MIOX4, has not been improved efficiently. In order to improve the activity of MIOX4, S. cerevisiae was used as a chassis microorganism in this study. The S. cerevisiae BY4741opi1Δ(BY4741 strain with knockout of OPI1 gene) was the starting strain in this experiment. Taking advantage of the fact that S. cerevisiae could use its own inositol-1-phosphate synthase(Ino1) to convert glucose-6-phosphate into inositol-1-phos-phate, which is then converted to inositol by its own inositol monophosphatase(Inm1/2), the inositol oxygenase miox4 gene from Arabidopsis thaliana and the uronate dehydrogenase udh gene from Pseudomonas syringae were exogenously expressed on the genome of BY4741opi1Δ strain to construct the glucaric acid metabolic pathway. Firstly, the udh gene was integrated into the terminator region of OPI1 of BY4741opi1Δ strain. Then the structure simulation of the inositol oxygenase MIOX4 from the Arabidopsis thaliana genome was performed to predict its catalytic active sites and ligand binding sites to identify the region of the key amino acids. The sequences of the key region were rando

关 键 词：酿酒酵母 肌醇加氧酶MIOX4 葡萄糖二酸 易错PCR 生物传感器 

分 类 号：TQ216[化学工程—有机化工]