构建多酶级联体系全细胞催化高效生物合成亚精胺

Efficient biosynthesis of spermidine via whole-cell catalysis with a multi-enzyme cascade system

摘要:

亚精胺是一种天然多胺，具有自噬诱导作用，能延长寿命、抑制肿瘤、保护心血管、调节神经及抗炎，在农业、工业和医药健康领域具有重要应用价值。本研究聚焦于利用微生物法合成亚精胺，通过追溯亚精胺前体物质的合成途径，过表达了前体物质合成的关键酶，在体外多酶级联条件下实现了以氨基酸为底物合成亚精胺，大大降低了生产成本。基于进化树挖掘筛选到酶活较高的枯草芽孢杆菌来源的S-腺苷甲硫氨酸脱羧酶，进一步通过理性设计对该酶进行定向进化改造，最终获得酶活性较野生型提高2.68倍的突变体。之后通过对比确认了全细胞催化合成亚精胺的优异性，并通过优化基因的串联顺序，引入ATP再生系统，实现了亚精胺的持续高效合成。在此基础上系统优化了反应体系，包括菌体量、底物浓度、反应温度、pH、金属离子，在最优反应条件下，重组大肠杆菌EC12在5 L反应器转化20 h，亚精胺最高产量达到17.83 g/L。本研究构建的重组大肠杆菌具有应用于工业生产亚精胺的潜力，为亚精胺的绿色高效合成奠定了理论和应用基础。

Abstract:

Spermidine is a natural polyamine with autophagy-inducing properties, capable of extending lifespan, inhibiting tumors, protecting cardiovascular health, regulating neural functions, and exerting anti-inflammatory effects. It holds significant applications in agriculture, industry, and healthcare. This study focuses on the microbial synthesis of spermidine by tracing the biosynthetic pathways of its precursors. Through the overexpression of key enzymes involved in precursor synthesis, spermidine was successfully synthesized in vitro with amino acids as substrates under multi-enzyme cascade conditions, which significantly reduced the production costs. The phylogenetic analysis identified an efficient S-adenosylmethionine decarboxylase from Bacillus subtilis, and its activity was enhanced by 2.68 folds through mutagenesis. Subsequently, the superiority of whole-cell catalysis for spermidine synthesis was confirmed, and the continuous and efficient synthesis of spermidine was achieved by optimizing gene tandem sequences and introducing an ATP regeneration system. Further, the reaction conditions, including cell density, substrate concentration, reaction temperature, pH, and metal ions, were optimized. Under optimal conditions, the recombinant Escherichia coli EC12 achieved a maximum spermidine yield of 17.83 g/L in a 5 L bioreactor after 20 hours of transformation. The recombinant E. coli constructed in this study demonstrates potential for industrial-scale spermidine production, laying a theoretical and practical foundation for the green and efficient synthesis of spermidine.