The Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
Appl Microbiol Biotechnol. 2018 Mar;102(5):2129-2141. doi: 10.1007/s00253-018-8741-y. Epub 2018 Jan 19.
Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD-dependent mutant formate dehydrogenase from Candida boidinii (CbFDH) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDH and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs leudh-3fdh was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs leudh-3fdh was 28.4 mg L min g dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L dry cells and 50.4 g L ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.
将两种或多种酶在单个宿主中共表达进行全细胞催化作为一种简单、低成本的生物合成方法已被广泛研究和应用，但几乎没有对多酶表达进行调控。在这里，我们通过在大肠杆菌中共同表达来自蜡状芽孢杆菌（Bacillus cereus）的亮氨酸脱氢酶（BcLeuDH）和来自粘质沙雷氏菌（Candida boidinii）的 NAD 依赖性突变型甲酸脱氢酶（CbFDH），开发了一种用于从苯甲酰甲酸生物合成 L-苯甘氨酸（L-Phg）的高效全细胞催化剂，具有可调节的多酶协调表达系统。通过共表达一个到四个拷贝的 CbFDH，并优化表达系统中 BcLeuDH 的 RBS 序列，最终将大肠杆菌 BL21/pETDuet-rbs leudh-3fdh 中的 BcLeuDH 与 CbFDH 的比例调节至 2:1，这是通过酶催化合成确定的最佳比例。使用全细胞转化进行 L-Phg 生产时，大肠杆菌 BL21/pETDuet-rbs leudh-3fdh 的催化剂活性为 28.4 mg L min g 干细胞重量，比没有酶表达调控的工程大肠杆菌高 3.7 倍。在最佳条件（pH 8.0 和 35°C）下，60 g L 的苯甲酰甲酸在 4.5 h 内完全转化为纯手性 L-Phg，使用 10 g L 干细胞和 50.4 g L 甲酸铵，对映体过量>99.9%。这种多酶协调表达系统策略显著提高了 L-Phg 的生产力，并为手性纯 L-Phg 的生产提供了一种新的低成本方法。
