代谢工程改造大肠杆菌生产尸胺

Metabolic engineering of Escherichia coli for the production of cadaverine

摘要:

尸胺是聚酰胺生产中的关键C5单体。由于细胞内5ʹ-磷酸吡哆醛(pyridoxal 5′-phosphate, PLP)再生效率有限，导致目前发酵法生产尸胺效率较低。本研究选择一株实验室保藏的赖氨酸高产大肠杆菌LY-4为研究对象，首先通过引入尸胺合成关键酶-赖氨酸脱羧酶(lysine decarboxylase, LDC)，成功构建了菌株L01，摇瓶发酵尸胺产量达1.07 g/L；随后开发了一种双代谢通路强化策略，协同增强内源和异源PLP合成模块，从而改善胞内PLP的合成，最优菌株L11摇瓶生产尸胺产量提升至9.23 g/L；最后在5 L发酵罐中对菌株L11生产尸胺的发酵工艺进行优化。工程菌株经48 h分批补料发酵，尸胺产量、得率、生产强度分别为54.43 g/L、0.22 g/g和1.13 g/(L·h)，具备一定的应用潜力。本研究可为构建包括尸胺在内的多种生物胺类细胞工厂提供理论依据和技术基础。

关键词:大肠杆菌;代谢工程;尸胺;5ʹ;-磷酸吡哆醛;发酵优化

Abstract:

Cadaverine is a fundamental C5 building block in the production of polyamides. Due to the limited regeneration efficiency of intracellular pyridoxal 5′-phosphate (PLP), the current fermentation-based production of cadaverine exhibits low efficiency. In this study, we developed an Escherichia coli strain L01 by introducing lysine decarboxylase (lysine decarboxylase, LDC, a key enzyme in the synthesis of cadaverine) into a lysine-producing strain E. coli LY-4, achieving a cadaverine tier of 1.07 g/L in shake flask fermentation. Subsequently, a dual metabolic pathway enhancement strategy was proposed to synergistically strengthen both endogenous and exogenous PLP synthesis modules, thereby improving intracellular PLP synthesis. The optimized strain L11 achieved a cadaverine titer of 9.23 g/L in shake flask fermentation. Finally, the fermentation process for cadaverine production by strain L11 was optimized in a 5 L fermenter. After 48 h of fed-batch fermentation, the engineered strain L11 achieved the cadaverine titer, yield, and productivity of 54.43 g/L, 0.22 g/g, and 1.13 g/(L·h), respectively. This study provides a theoretical and technical foundation for establishing microbial cell factories for bioamine production.

Key words:Escherichia coli;metabolic engineering;cadaverine;pyridoxal 5′-phosphate;fermentation optimization