Abstract
Streptomycetes are key producers of natural products (NPs), and engineering these organisms represents an effective strategy for enhancing NPs production. However, genetic elements (e.g., promoters and ribosome-binding sites (RBSs)) developed in wild-type or well-characterized model strains often exhibit limited adaptability in industrial strains, hindering their broader application. In this study, an efficient, low-cost strategy for constructing a gene expression element library (GEEL) for industrial Streptomyces was developed and validated using the ε-poly-ʟ-lysine (ε-PL) producing strain S. albulus GS114. Firstly, commonly used reporter genes were screened, and β-glucuronidase (gusA) was selected as the optimal reporter. Subsequently, a mixed ligation system (MLS) based on Gibson assembly was established to enable efficient assembly of pooled fragments into a vector. Using this system, libraries comprising 60 promoters and 30 RBSs were constructed. Each library was then introduced separately into S. albulus GS114 by single-round conjugation, and the resulting conjugants were screened for GUS activity. From these screens, 30 promoters and 14 RBSs spanning a range of strengths were identified, yielding GEELs specifically tailored to S. albulus GS114. Thirdly, five promoters and three RBSs were selected to optimize the expression of the pls gene, resulting in an ε-PL production of 4.73 g/L, reflecting a 2.13-fold increase compared to the control. Finally, this strategy was similarly applied to S. gilvosporeus to establish GEELs and optimize the expression of sgnM, leading to enhanced natamycin production. The customized GEEL construction strategy developed in this study provides a foundational toolkit for the precise regulation of key targets in industrial Streptomyces and the further enhancement of NPs biosynthesis.