Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Bioorg Med Chem. 2020 May 15;28(10):115464. doi: 10.1016/j.bmc.2020.115464. Epub 2020 Mar 29.
A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor d-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and d-glyceraldehyde was performed by l-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars d-sorbose and d-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldO) was expressed in E. coli and the purified AldO was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldO was constructed. Under the optimized conditions, it produced 7.9 g/L of d-sorbose and d-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.
建立了一种使用大肠杆菌全细胞进行级联合成稀有糖的合成平台。在级联反应中，供体底物二羟丙酮磷酸（DHAP）由甘油激酶（GK）和甘油磷酸氧化酶（GPO）从甘油中生成。受体 d-甘油醛可直接由甘油通过糖醇氧化酶产生。然后，l-鼠李糖-1-磷酸醛缩酶（RhaD）使 DHAP 和 d-甘油醛之间发生醛醇缩合反应，生成相应的糖-1-磷酸。最后，通过果糖-1-磷酸酶（YqaB）去除磷酸基团，得到稀有糖 d-山梨糖和 d-阿洛酮糖。为了实现这一目标，在大肠杆菌中表达了来自草色链霉菌（AldO）的糖醇氧化酶，并对纯化的 AldO 进行了表征。此外，构建了一株过表达包括 AldO 在内的六种酶的重组大肠杆菌菌株。在优化条件下，从甘油生产 7.9 g/L 的 d-山梨糖和 d-阿洛酮糖，总转化率为 17.7%。本研究为稀有糖的合成提供了一种有用且经济有效的方法。
