Construction of a library of gene expression elements in industrial Streptomyces and its application

<i>Streptomycetes</i> 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 <i>Streptomyces</i> was developed and validated using the ε-poly-ʟ-lysine (ε-PL) producing strain <i>S. albulus</i> GS114. Firstly, commonly used reporter genes were screened, and β-glucuronidase (<i>gusA</i>) 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 <i>S. albulus</i> 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 <i>S. albulus</i> GS114. Thirdly, five promoters and three RBSs were selected to optimize the expression of the <i>pls</i> 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 <i>S. gilvosporeus</i> to establish GEELs and optimize the expression of <i>sgnM</i>, 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 <i>Streptomyces</i> and the further enhancement of NPs biosynthesis.