代谢工程改造大肠杆菌生产乙醇酸

Metabolic engineering of Escherichia coli for glycolic acid production

摘要:

乙醇酸是结构最简单的α-羟基酸，广泛应用于医药、化工等领域。然而，目前发酵生产乙醇酸的菌株普遍含有质粒或需要添加诱导剂，不利于大规模工业生产。为了构建无需质粒和化学诱导剂的乙醇酸生产菌株，本研究选择野生型大肠杆菌ATCC 8739作为出发菌株，首先筛选了不同来源的异柠檬酸裂解酶和乙醛酸还原酶，并通过基因组水平整合获得了工程菌株GA01，罐上发酵测试该菌株的乙醇酸产量达到10.12 g/L，经过路径酶拷贝数优化后产量进一步提升至15.25 g/L；其次，通过阻断乙醇酸合成途径的分支代谢路径并强化前体供应，将碳代谢流高效引入乙醇酸合成途径，最优菌株GA07-1的乙醇酸产量提高至41.69 g/L；最后，在5 L发酵罐中进行发酵优化，菌株GA07-1的乙醇酸产量、葡萄糖得率和生产强度分别达到50.62 g/L，0.49 g/g和1.20 g/(h·L)。本研究实现了在无抗生素和化学诱导剂条件下的乙醇酸从头发酵。

Abstract:

Glycolic acid, the simplest α-hydroxy acid in structure, is extensively utilized in pharmaceuticals, chemicals, and other fields. However, the strains currently employed for the production of glycolic acid by fermentation typically possess plasmids or necessitate the addition of chemical inducers, which hampers large-scale industrial production. In order to construct a glycolic acid producing strain that does not require plasmids or chemical inducers. In this study, we utilized the wild-type strain Escherichia coli ATCC 8739 as the starting strain. Initially, we screened isocitrate lyase and glyoxylate reductase from different sources and constructed an engineered strain GA01 by genomic integration. Fermentation with this strain in a fermenter produced glycolic acid at a titer of 10.12 g/L. Following optimization of the copy number of pathway enzymes, the titer of glycolic acid was further increased to 15.25 g/L. Additionally, by blocking the branched metabolic pathways in the glycolic acid synthesis route and enhancing precursor supply, we effectively redirected carbon flow towards glycolic acid production, and fermentation with the optimized strain GA07-1 achieved the glycolic acid titer of 41.69 g/L. Finally, fermentation optimization was carried out with strain GA07-1 in a 5 L fermenter, achieving a glycolic acid titer of 50.62 g/L, a yield of 0.49 g/g, and productivity of 1.20 g/(h·L). These results realized the de novo fermentation of glycolic acid from glucose without antibiotics and chemical inducers.