The PreKit platform: Cryptic gene clusters activation and high-titer compound production

The discovery of novel scaffold compounds or enzymes through genome mining of biosynthetic gene clusters (BGCs) represents cornerstone of drug development. A major obstacle, however, is that most microbial BGCs encoding secondary metabolites remain silent under standard laboratory conditions. While promoter engineering coupled with heterologous expression has become a mainstream strategy to activate these silent BGCs, its efficiency is often limited by inherent incompatibilities among key factors, including host selection, promoter strength, and culture medium. To overcome this issue, we developed a streamlined platform integrating a promoter-library with an indC-based reporter system. This platform enables combinatorial screening across diverse media conditions and different heterologous hosts, systematically resolving the multi-factor incompatibility issue. Building on this strategy, we further developed a plug-and-play promoter toolkit, which was successfully applied to activate a silent type II polyketide synthase (PKS) gene cluster (spa). This led to the production of one new aromatic polyketides compounds strepanthenes A (1a) and streptoketide C (1b). Furthermore, enhancement of precursor supply was achieved by integrating a non-carboxylative malonyl-CoA (NCM) pathway into S. lividans LJ1018 through att P ^TG1 site-specific integration system, we achieved a 3.6-fold and 3.3-fold increase in the titers of compounds 1a and 1b, respectively. Collectively, this work provides a versatile and efficient strategy for activating silent BGCs and improving metabolite production. Bioactivity evaluation revealed that new compound 1a exhibited antibacterial activity against S. aureus and E. faecalis, with a consistent MIC value of 4 μg mL^-1 against both strains.