Identification and Combinatorial Overexpression of Key Genes for Enhancing ε-Poly-L-lysine Biosynthesis in <i>Streptomyces albulus</i>

ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative mainly produced by the aerobic, filamentous bacterium <i>Streptomyces albulus</i>. Therefore, it is crucial to breed superior ε-PL-producing strains to enhance fermentation efficiency to reduce production costs. Metabolic engineering is an effective measure for strain modification, but there are few reports on key genes for ε-PL biosynthesis. In this study, metabolic flux analysis was employed to identify potential key genes in ε-PL biosynthesis in <i>S. albulus</i> WG-608. A total of six potential key genes were identified. Three effective key genes (<i>ppc</i>, <i>pyc</i> and <i>pls</i>) were identified for the first time in ε-PL biosynthesis through overexpression experiments. It also presents the first demonstration of the promoting effects of <i>ppc</i> and <i>pyc</i> on ε-PL biosynthesis. Three genes were then co-expressed in <i>S. albulus</i> WG-608 to obtain OE-<i>ppc-pyc-pls</i>, which exhibited an 11.4% increase in ε-PL production compared to <i>S. albulus</i> WG-608, with a 25.5% increase in specific ε-PL production. Finally, the metabolic flux analysis of OE-<i>ppc-pyc-pls</i> compared to <i>S. albulus</i> WG-608 demonstrated that OE-<i>ppc-pyc-pls</i> successfully altered the metabolic flux as expected. This study not only provides a theoretical basis for the metabolic engineering of ε-PL-producing strains but also provides an effective approach for the metabolic engineering of other metabolites.