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 randomly mutated by two rounds of error-prone PCR and the mutated miox4* was integrated into the promoter region of OPI1 of BY4741opi1Δ strain to construct its mutant library. Taking advantage of the fact that the E. coli glucaric acid biosensor plasmid of R7M10, expressing both transcription activator CdaR and reporter gene green fluorescent protein GFP, can display the concentration of glucaric acid as fluorescence intensity, it was used as a high-throughput screening tool to screen the MIOX4 mutant library. As a result, 60 strains with higher fluorescence were obtained from the preliminary screen. Subsequently, they were further verified by shake flask fermentation and 24 strains were obtained whose glucaric acid production were higher than the control. Sequencing analysis showed that the four potential MIOX4 mutants (S133R, K88R, E72V, T40P) were obtained from the mutant library which increased the production of glucaric acid by 53%, 30%, 21%, and 17% compared with the control strains, respectively. Finally, by analyzing the protein structures of the mutant MIOX4 (S133R) with the highest increase in glucaric acid production and wild type MIOX4, it was found that the mutation site R133 increased the steric distance between the site and the substrate inositol, which reduced the steric hindrance and thus increased its catalytic ability of inositol. This study further proves that MIOX4 plays an important role in the biosynthesis of glucaric acid and will provide foundation for the subsequent research on the structure and function of MIOX and the further improvement of glucaric acid production.
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