代谢工程改造大肠杆菌生产四碳二羧酸

Metabolic engineering of Escherichia coli for the production of four-carbon dicarboxylic acids

摘要:

目的 四碳二羧酸是一类重要的平台化学品，在食品、医药及化工领域应用广泛。然而，微生物发酵法生产四碳二羧酸的效率仍面临挑战，主要受限于中心碳代谢通量不足和副产物积累等问题。方法 以大肠杆菌(Escherichia coli)为底盘菌株，采用理性代谢工程与非理性改造相结合的策略，系统优化E. coli的四碳二羧酸合成能力。结果 通过重构非循环乙醛酸支路、优化路径关键酶的表达水平，提高了四碳二羧酸代谢通量；借助常压室温等离子体诱变(atmospheric and room-temperature plasma, ARTP)技术提升了四碳二羧酸的合成能力。敲除乙酸、甲酸和乳酸代谢途径的相关基因有效减少了碳代谢流的损耗，提高了四碳二羧酸合成前体草酰乙酸的供给效率。进一步通过特异性改造终端代谢路径构建了生产富马酸的工程菌株E. coli Fum02。在5 L发酵罐中，工程菌株E. coli Fum02的富马酸产量、得率和生产强度分别达到45.2 g/L、0.45 g/g和0.23 g/(L·h)。在此基础上，通过阻断琥珀酸脱氢酶基因(sdhAB)和发酵优化等策略可进一步用于生产琥珀酸。结论 本研究为代谢工程改造细菌生产有机酸提供了参考，同时也为四碳二羧酸的工业化生物制造奠定了基础。

Abstract:

Objective Four-carbon dicarboxylic acids are a class of important platform chemicals widely used in the food, pharmaceutical, and chemical industries. However, the efficiency of microbial fermentation for producing four-carbon dicarboxylic acids still faces challenges, mainly limited by insufficient central carbon metabolic flux and byproduct accumulation.Methods This study used Escherichia coli as the chassis strain and adopted a strategy combining rational metabolic engineering and non-rational modification to systematically optimize the four-carbon dicarboxylic acid synthesis capacity of E. coli.Results The non-cyclic glyoxylate shunt was reconstructed and the expression of key pathway enzymes was optimized to enhance the metabolic flux toward four-carbon dicarboxylic acids. The synthesis capacity of four-carbon dicarboxylic acids was enhanced by employing atmospheric and room-temperature plasma (ARTP) mutagenesis. The knockout of key genes in the acetate, formate, and lactate synthesis pathways effectively minimized carbon flux diversion, thereby enhancing the availability of oxaloacetate, the central precursor to four-carbon dicarboxylic acids. On this basis, through specific modification of terminal metabolic pathways, the engineering strain E. coli Fum02 for fumaric acid production were constructed. Finally, in a 5 L fermenter, the fumaric acid titer, yield, and productivity of the engineering strain E. coli Fum02 reached 45.2 g/L, 0.45 g/g, and 0.23 g/(L·h), respectively. Furthermore, by blocking the succinate dehydrogenase gene (sdhAB) and implementing fermentation optimization strategies, this platform strain could also be redirected toward efficient succinate production.Conclusion This study provides a reference for the metabolic engineering modification of bacteria to produce organic acids and also lays a foundation for the industrial biomanufacturing of four-carbon dicarboxylic acids.