代谢工程改造大肠杆菌生产α-酮戊二酸

Metabolic engineering of Escherichia coli for α-ketoglutarate production

摘要:

α-酮戊二酸是一种重要的短链有机酸，广泛应用于食品、医药、化妆品和饲料等领域。然而，生物发酵法生产α-酮戊二酸的效率有待进一步提高，主要受限于微生物内源代谢途径的合成能力。目的 开发能高效生产α-酮戊二酸的大肠杆菌，为未来大规模生产α-酮戊二酸提供理论支撑。方法 采用理性代谢工程改造α-酮戊二酸合成途径与非理性常压室温等离子体诱变(atmospheric and room temperature plasma mutagenesis, ARTP)相结合的方法解除内源代谢途径的限制，提高α-酮戊二酸的合成效率。结果 通过表达丙酮酸羧化酶、柠檬酸合成酶、顺乌头酸酶和异柠檬酸脱氢酶重构了α-酮戊二酸合成的氧化TCA途径，有效提升了α-酮戊二酸产量。借助ARTP技术，非理性优化并强化了α-酮戊二酸合成的代谢网络，进一步提高了其合成能力。通过敲除乳酸、乙酸和甲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α-酮戊二酸合成前体——丙酮酸的供给效率。在此基础上，进一步敲除α-酮戊二酸降解途径的相关基因，使碳代谢流在α-酮戊二酸节点滞留，提高了α-酮戊二酸产量。通过优化发酵条件，在5 L发酵罐中工程菌株Escherichia coli KA29的α-酮戊二酸产量、得率和生产强度分别达到28.7 g/L、0.29 g/g和0.48 g/(L?h)。结论 本研究策略为α-酮戊二酸高产菌株的开发与应用奠定了基础，同时也为代谢工程改造生产其他有机酸提供了参考。

Abstract:

α-ketoglutarate is an important short-chain organic acid that is widely used in various fields such as food, medicine, cosmetics, and animal feed. However, the efficiency of producing α-ketoglutarate through biological fermentation remains to be improved, primarily due to the limitations in the synthetic capacity of microbial metabolic pathways.Objective To address the above issues, we developed an engineered Escherichia coli that can efficiently produce α-ketoglutarate, thereby providing theoretical support for the large-scale production of α-ketoglutarate in the future.Methods We employed an efficient approach combining rational and irrational modifications to overcome the constraints of endogenous metabolic pathways and enhance the biosynthesis efficiency of α-ketoglutarate.Results The oxidative TCA pathway was reconstructed to improve α-ketoglutarate production through expressing pyruvate carboxylase, citrate synthase, aconitase, and isocitrate dehydrogenase. The metabolic network for α-ketoglutarate biosynthesis was irrationally optimized and strengthened to enhance its biosynthesis capability by atmospheric pressure room temperature plasma mutagenesis. To improve the supply efficiency of the precursor for α-ketoglutarate biosynthesis, we reduced the dissipation of carbon flux in the pyruvate node by knocking out genes related to the accumulation of lactate, acetate, and formate. Furthermore, we knocked out the genes related to the degradation pathway of α-ketoglutarate to achieve the retention of carbon flux at α-ketoglutarate node and improve its production. Through the optimization of fermentation conditions, the fermentation in a 5 L fermenter with the engineered strain E. coli KA29 achieved the α-ketoglutarate titer, yield, and productivity of 28.7 g/L, 0.29 g/g, and 0.48 g/(L·h), respectively.Conclusion The research strategies mentioned above lay a foundation for the development and application of strains with high production of α-ketoglutarate and provide a reference for metabolic engineering to produce other organic acids.