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

Area of Science:

• Synthetic biology
• Natural product drug discovery
• Metabolic engineering

Background:

• Genome mining of biosynthetic gene clusters (BGCs) is crucial for identifying novel drug scaffolds.
• Many microbial BGCs remain silent under laboratory conditions, hindering natural product discovery.
• Activating silent BGCs via promoter engineering and heterologous expression faces challenges like host-promoter-media incompatibilities.

Purpose of the Study:

• To develop a streamlined platform for activating silent BGCs by overcoming multi-factor incompatibilities.
• To create a plug-and-play promoter toolkit for efficient BGC activation.
• To enhance the production of novel secondary metabolites and evaluate their bioactivity.

Main Methods:

• Developed a platform integrating a promoter library with an indC-based reporter system for combinatorial screening.
• Applied the platform and a plug-and-play promoter toolkit to activate a silent type II polyketide synthase (PKS) gene cluster (spa).
• Integrated a non-carboxylative malonyl-CoA (NCM) pathway into S. lividans to enhance precursor supply.

Main Results:

• Successfully activated the silent spa BGC, yielding new aromatic polyketides, strepanthenes A (1a) and streptoketide C (1b).
• Achieved a 3.6-fold and 3.3-fold increase in titers for compounds 1a and 1b, respectively, after NCM pathway integration.
• Compound 1a demonstrated antibacterial activity against S. aureus and E. faecalis (MIC: 4 μg mL^-1).

Conclusions:

• The developed platform and toolkit offer a versatile and efficient strategy for activating silent BGCs.
• This approach significantly improves secondary metabolite production through combinatorial screening and precursor enhancement.
• The discovery of bioactive compound 1a highlights the potential of this method for novel drug discovery.