Highly efficient spermidine production system in Escherichia coli BL21 (DE3) based on precursor metabolic modules...

Spermidine, a naturally occurring polyamine compound, has garnered significant attention due to its versatile physiological functions, including induction of cellular autophagy, antioxidant activity, and maintenance of mitochondrial homeostasis. In this study, we established a novel spermidine biosynthesis system in E. coli BL21 (DE3) by heterologously introducing the carboxyaminopropylagmatine (CAPA) pathway derived from cyanobacterium. To enhance precursor supply, we overexpressed key enzymes in the L-aspartate β-semialdehyde and agmatine branch metabolic pathways within the precursor metabolic module, while simultaneously knocking out competing metabolic pathways to redirect metabolic flux toward spermidine biosynthesis. To address the challenge of intracellular spermidine accumulation and its associated cytotoxicity, the high-efficiency spermidine efflux pump protein AmvA from Acinetobacter baumannii was heterologously expressed in E. coli BL21 (DE3). This engineering strategy enabled efficient extrusion of spermidine from the cells, alleviating the toxic effects of high intracellular spermidine concentrations on the host strain. Through these metabolic and efflux pump engineering modifications, the engineered strain SPD06-P5-P6 was constructed. Following 48 h of shake flask fermentation, SPD06-P5-P6 achieved a spermidine titer of 163.11 mg/L, which further increased to 1164.22 mg/L after 96 h of scale-up cultivation in a 5 L bioreactor.