University of Chinese Academy of Sciences, Beijing, 100049, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; College of Biotechnology, Tianjin University of Sciences and Technology, Tianjin, 300457, China.
Metab Eng. 2020 Sep;61:152-159. doi: 10.1016/j.ymben.2020.06.001. Epub 2020 Jun 10.
Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO. In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value.
乙醇酸盐是一种大宗化学品，广泛应用于纺织、食品加工和制药行业。乙醇酸盐可以通过微生物发酵从糖中生产。然而，当使用葡萄糖作为唯一的碳源时，由于 CO 的损失，乙醇酸盐的理论最大碳摩尔产率为 0.67 mol/mol。在这项研究中，设计了一种协同系统来同时利用乙酸盐和葡萄糖，以提高碳产率。葡萄糖的主要功能是提供 NADPH，而乙酸盐则提供生产乙醇酸盐的主要碳骨架。理论上，1 葡萄糖和 5 乙酸盐可以生产 6 乙醇酸盐，碳摩尔产率可以提高到 0.75 mol/mol。整个合成途径分为两个模块，一个用于将乙酸盐转化为乙醇酸盐，另一个用于利用葡萄糖提供 NADPH。通过激活 acs、gltA、aceA 和 ycdW 对模块 I 进行工程改造后，乙醇酸盐的浓度从 0.07 提高到 2.16 g/L，而乙醇酸盐的产率从 0.04 提高到 0.35 mol/mol-乙酸盐和从 0.03 提高到 1.04 mol/mol-葡萄糖。然后对模块 II 进行工程改造以增加 NADPH 的供应。通过删除 pfkA、pfkB、ptsI 和 sthA 基因以及上调 zwf、pgl 和 tktA，乙醇酸盐的浓度从 2.16 提高到 4.86 g/L，而乙醇酸盐的产率从 0.35 提高到 0.82 mol/mol-乙酸盐和从 1.04 提高到 6.03 mol/mol-葡萄糖。进一步提高 AceA 和 YcdW 的活性以将碳通量拉向乙醇酸盐，将乙醇酸盐的产率从 0.82 提高到 0.92 mol/mol-乙酸盐。最终菌株 NZ-Gly303 的分批补料发酵生产了 73.3 g/L 的乙醇酸盐，比生产率为 1.04 g/(L·h)。乙酸盐到乙醇酸盐的产率为 0.85 mol/mol（1.08 g/g），而葡萄糖到乙醇酸盐的产率为 6.1 mol/mol（2.58 g/g）。总碳摩尔产率为 0.60 mol/mol，达到理论值的 80%。
