基于代谢工程和多酶级联策略促进大肠杆菌合成水杨酸

Promoting the Synthesis of Salicylic Acid in Escherichia coli Based on Metabolic Engineering and Multi-enzyme Cascade Strategy

摘要:

【目的】芳香族化合物水杨酸（SA）的产量受限于其对细胞能量代谢相关酶的抑制作用，为避免SA对细胞能量代谢相关酶的影响，需提升SA产量。【方法】通过优化代谢途径和酶系统，设计了高效的大肠杆菌（Escherichia coli）菌株来提高SA产量。【结果】根据GEMS1515基因组规模的代谢模型，预测了磷酸烯醇式丙酮酸（PEP）途径和SA生物合成途径中的关键瓶颈，并通过靶向敲除fbaA和dcuA基因，成功将SA产量从834.82 mg/L提升至1 143.56 mg/L。通过结合柔性和刚性肽接头以及TRAP支架蛋白质（TRAP1、TRAP3）的多酶级联策略，提高了催化效率，最终优化菌株的SA产量最高达2 100.00 mg/L，比初始菌株提高了151%。【结论】将代谢工程、多酶级联策略和机器学习技术运用在高价值化合物的生物合成优化中具有较大应用潜力。

Abstract:

[Objective] The production of the aromatic compound salicylic acid (SA) is limited by its inhibitory effects on enzymes related to cellular energy metabolism. This study aims to develop a strategy for avoiding the impact of SA on enzymes related to cellular energy metabolism and increase the yield of SA.[Method] A highly efficient Escherichia coli strain was designed to improve SA production by optimizing metabolic pathways and enzyme systems.[Result] The genome-scale metabolic model GEMS1515 was employed to predict the key bottle necks in the phosphoenolpyruvic acid (PEP) pathway and SA biosynthesis pathways. The SA yield was successfully increased from 834.82 mg/L to 1 143.56 mg/L by targeted knockout of fbaA and dcuA genes. Furthermore, the enzyme fusion strategy, combining flexible and rigid peptide linkers with TRAP scaffold proteins (TRAP1 and TRAP3), further improved the catalytic efficiency. The optimized strain achieved a maximum SA yield of 2 100.00 mg/L, which represented an increase of 151% over that of the initial strain.[Conclusion] Integrating metabolic engineering, multi-enzyme cascade strategy, and machine learning demonstrate great application potential in optimizing the biosynthesis of high-value compounds.