Controlled chain length production of ε-poly-l-lysine by Streptomyces albulus

ε-Poly- l -lysine (ε-PL), typically comprising 25–35  l -lysine residues, is a natural homopolymer with potent antibacterial properties. Its wider use is limited by two key challenges: precise control over peptide chain length and low production yields of low-molecular-weight (low-MW) ε-PL variants. In this study, we addressed these constraints by combining genetic engineering, carbon source modulation, and fermentation optimisation, enabling the targeted production of four well-characterised ε-PL variants. Multiple genetically engineered strains of Streptomyces albulus were developed and evaluated for ε-PL chain distribution. Particularly, S. albulus CICC-Pls880-LysP— co -overexpression of a mutated polylysine synthetase (Pls S880I) and a lysine importer (LysP)—produced ε-PL with chain lengths between 23 and 31 residues when grown in glucose-enriched fermentation broth and between 19 and 30 residues in glycerol-enriched broth. By contrast, the parental strain CICC-Pls-LysP yielded longer polymers (28–34 residues in glucose and 25–32 in glycerol), confirming the effectiveness of enzyme engineering in modulating chain length. Subsequent statistical optimisation of the fermentation medium, coupled with fed-batch cultivation strategies, enabled all four ε-PL variants (28–34, 25–32, 23–31, and 19–30 mer) to achieve production titres exceeding 50 g/L. Notably, supplementing l -lysine during fermentation significantly enhanced the yield of the low-MW variant (23–31 mer), reaching to 75.35 g/L. Antibacterial activity assays revealed a distinct correlation between chain length and efficacy, providing valuable insights into the structure–function relationships of ε-PL. This method, established for selectively synthesising ε-PL variants, overcomes historical yield bottlenecks for low-MW ε-PL, thereby broadening the applications of ε-PL.