Engineering Escherichia coli for high-titer de novo biosynthesis of the N-nucleoside antibiotic Angustmycin A

Abstract Background Angustmycin A (also known as guvermectin) is a rare N-nucleoside antibiotic with broad-spectrum antimicrobial activity and registered agricultural use as a plant growth-promoting biochemical pesticide. Native Streptomyces producers remain the primary production source, but their slow growth, long fermentation cycles, and limited genetic tractability constrain both titer improvement and process development. Establishing a fast-growing, genetically flexible host for de novo Angustmycin A biosynthesis from glucose is therefore a practical priority. Results An Escherichia coli ( E. coli ) microbial cell factory was constructed for de novo Angustmycin A biosynthesis through four sequential engineering stages. First, consolidation of the six-enzyme pathway onto a single medium-copy plasmid and deletion of pfkA to redirect fructose-6-phosphate (F6P) flux increased production from 142 mg L − 1 to 649 ± 9.0 mg L − 1 . Second, pathway bottleneck analysis identified AgmC solubility as a primary constraint; AgmC-centered liquid–liquid phase separation (LLPS) recruitment, combined with co-recruitment of AgmA, relieved this bottleneck and improved production to 699 ± 7.2 mg L − 1 . Third, orthogonal optimization of cultivation parameters and chromosomal gltA 5′-UTR engineering to moderate TCA cycle flux further increased shake-flask production to 1225 ± 12.9 mg L − 1 . Finally, fed-batch fermentation of the final strain in a 1.2-L bioreactor produced 1805 ± 74.1 mg L − 1 Angustmycin A at 96 h (volumetric productivity: ~18.8 mg L −1 h − 1 ). Conclusions This work establishes a high-performing E. coli cell factory for gram-per-liter de novo production of Angustmycin A from glucose. The engineered strain reached 1.805 g L − 1 within 96 h and a maximum titer of approximately 1.92 g L − 1 during extended fed-batch cultivation, demonstrating rapid product accumulation while achieving the highest reported Angustmycin A titer to date. F6P flux rewiring and mitigation of limited soluble AgmC availability emerged as two major engineering levers for improving pathway performance. More broadly, the integrated strategy of precursor-flux redistribution and bottleneck-guided enzyme recruitment provides a transferable framework for microbial production of related N-nucleoside natural products.