高效合成葡萄糖二酸酿酒酵母工程菌的构建

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid

摘要:

葡萄糖二酸是天然存在的一种重要二元酸，其在医疗保健和化工工业等领域具有很高的实际应用价值，因此被称为“最具价值的生物炼制产品之一”。以酿酒酵母（Saccharomyces cerevisiae）为底盘微生物，文中考察了过量表达肌醇转运蛋白Itr1、融合表达肌醇加氧酶和葡萄糖醛酸脱氢酶以及弱化表达葡萄糖6-磷酸脱氢酶基因ZWF1三种策略对葡萄糖二酸产量的影响。研究结果显示，过量表达肌醇转运蛋白Itr1使葡萄糖二酸产量在摇瓶发酵条件下较出发菌株Bga-3提高了26%；MIOX4-Udh融合蛋白的表达使葡萄糖二酸的产量较Bga-3菌株提高了40%；在此基础上，弱化表达葡萄糖6-磷酸脱氢酶基因ZWF1后，葡萄糖二酸的产量达5.5 g/L，较相同发酵条件下Bga-3菌株提高了60%。在5 L发酵罐中，该菌株葡萄糖二酸的最高产量达10.85 g/L，较Bga-3菌株提高了80%。由此可见，上述代谢改造策略的应用在很大程度上提高了葡萄糖二酸的途径效率和产量，为通过代谢工程方法在酿酒酵母中合成其他化合物的研究提供了参考。

Abstract:

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.