D-glycerate is an attractive chemical for a wide variety of pharmaceutical, cosmetic, biodegradable polymers, and other applications. Now several studies have been reported about the synthesis of glycerate by different biotechnological and chemical routes from glycerol or other feedstock. Here, we present the construction of an Escherichia coli engineered strain to produce optically pure D-glycerate by oxidizing glycerol with an evolved variant of alditol oxidase (AldO) from Streptomyces coelicolor. This is achieved by starting from a previously reported variant mAldO and employing three rounds of directed evolution, as well as the combination of growth-coupled high throughput selection with colorimetric screening. The variant eAldO3-24 displays a higher substrate affinity toward glycerol with 5.23-fold than the wild-type AldO, and a 1.85-fold increase of catalytic efficiency (kcat/KM). Then we introduced an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 expression system in E. coli to overexpress the variant eAldO3-24, and deleted glucosylglycerate phosphorylase encoding gene ycjM to block the consumption of D-glycerate. Finally, the resulting strain TZ-170 produced 30.1 g/l D-glycerate at 70 h with a yield of 0.376 mol/mol in 5-l fed-batch fermentation.

中文翻译：

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糖醇氧化酶的定向进化，用于从工程大肠杆菌中的甘油生产光学纯 D-甘油酸酯

D-甘油酸酯是一种有吸引力的化学品，适用于各种药物、化妆品、可生物降解聚合物和其他应用。现在已经报道了几项关于通过不同的生物技术和化学途径从甘油或其他原料合成甘油酸酯的研究。在这里，我们提出了一种大肠杆菌工程菌株的构建，该菌株通过用来自天蓝色链霉菌的糖醇氧化酶 (AldO) 的进化变体氧化甘油来生产光学纯的 D-甘油酸。这是通过从先前报道的变体 mAldO 开始并采用三轮定向进化以及将生长耦合的高通量选择与比色筛选相结合来实现的。变体 eAldO3-24 对甘油的底物亲和力比野生型 AldO 高 5.23 倍，为 1。催化效率提高 85 倍 (kcat/KM)。然后我们在大肠杆菌中引入异丙基-β-D-硫代吡喃半乳糖苷 (IPTG) 诱导的 T7 表达系统，以过表达变体 eAldO3-24，并删除葡萄糖基甘油酸磷酸化酶编码基因 ycjM 以阻断 D-甘油酸的消耗。最后，所得菌株 TZ-170 在 5 升补料分批发酵中 70 小时产生 30.1 g/l D-甘油酸，产量为 0.376 mol/mol。