School of Chemical and Biomolecular Engineering, Pusan National University, Busan, South Korea; School of Energy and Chemical Engineering, UNIST, Ulsan, South Korea.
School of Chemical and Biomolecular Engineering, Pusan National University, Busan, South Korea.
Metab Eng. 2020 Nov;62:116-125. doi: 10.1016/j.ymben.2020.09.001. Epub 2020 Sep 6.
1,3-Propanediol (1,3-PDO) is an important platform chemical which has a wide application in food, cosmetics, pharmaceutical and textile industries. Its biological production using recombinant Escherichia coli with glucose as carbon source has been commercialized by DuPont, but E. coli cannot synthesize coenzyme B which is an essential and expensive cofactor of glycerol dehydratase, a core enzyme in 1,3-PDO biosynthesis. This study aims to develop a more economical microbial cell factory using Klebsiella pneumoniae J2B which can naturally synthesize coenzyme B. To this end, the heterologous pathway for the production of glycerol from dihydroxyacetone-3-phosphate (DHAP), a glycolytic intermediate, was introduced to J2B and, afterwards, the strain was extensively modified for carbon and energy metabolisms including: (i) removal of carbon catabolite repression, (ii) blockage of glycerol export across the cell membrane, (iii) improvement of NADH regeneration/availability, (iv) modification of TCA cycle and electron transport chain, (v) overexpression of 1,3-PDO module enzyme, and (vi) overexpression of glucose transporter. A total of 33 genes were modified and/or overexpressed, and one resulting strain could produce 814 mM (62 g/L) of 1,3-PDO with the yield of 1.27 mol/mol glucose in fed-batch bioreactor culture with a limited supplementation of coenzyme B at 4 μM, which is ~10 fold less than that employed by DuPont. This study highlights the importance of balanced use of glucose in the production of carbon backbone of the target chemical (1,3-PDO) and regeneration of reducing power (NADH). This study also suggests that K. pneumoniae J2B is a promising host for the production of 1,3-PDO from glucose.
1,3-丙二醇（1,3-PDO）是一种重要的平台化学品，在食品、化妆品、制药和纺织工业中有广泛的应用。其以葡萄糖为碳源的生物生产已被杜邦公司商业化，但大肠杆菌不能合成辅酶 B，而辅酶 B 是甘油脱水酶（1,3-PDO 生物合成的核心酶）所必需的昂贵辅因子。本研究旨在开发一种更经济的微生物细胞工厂，使用能够自然合成辅酶 B 的肺炎克雷伯氏菌 J2B。为此，引入了一条从二羟丙酮-3-磷酸（DHAP）生产甘油的异源途径，DHAP 是糖酵解的中间产物，然后对 J2B 进行了广泛的碳和能量代谢改造，包括：（i）去除碳分解代谢物阻遏，（ii）阻断甘油穿过细胞膜的出口，（iii）改善 NADH 的再生/可用性，（iv）修饰 TCA 循环和电子传递链，（v）过表达 1,3-PDO 模块酶，和（vi）过表达葡萄糖转运蛋白。共修饰和/或过表达了 33 个基因，其中一个菌株在补加 4 μM 辅酶 B 的限制条件下，在分批补料生物反应器培养中可生产 814 mM（62 g/L）的 1,3-PDO，其得率为 1.27 mol/mol 葡萄糖，这比杜邦公司使用的量少了约 10 倍。本研究强调了在目标化学品（1,3-PDO）的碳骨架生产和还原力（NADH）再生中平衡使用葡萄糖的重要性。本研究还表明，肺炎克雷伯氏菌 J2B 是从葡萄糖生产 1,3-PDO 的有前途的宿主。
