A high-throughput chemogenetic screening platform for activating silent biosynthetic gene clusters to discover new metabolites

Background

Natural products derived from microorganisms, particularly actinomycete bacteria, have historically served as a rich source of clinically valuable compounds, including antibiotics and anticancer agents. Advances in genome sequencing have revealed that these bacteria harbor a vast array of biosynthetic gene clusters (BGCs) with the potential to produce novel secondary metabolites. However, the majority of these BGCs are “silent” or “cryptic” under standard laboratory conditions, meaning they are not naturally expressed and their chemical products remain unknown. Unlocking these hidden pathways is critical for expanding the chemical diversity available for drug discovery, especially in the face of rising antibiotic resistance and the ongoing need for new therapeutics.

Despite the promise of genome mining, current approaches to activating silent BGCs are limited and often inefficient. Traditional methods, such as altering growth conditions or random mutagenesis, are labor-intensive, non-specific, and frequently fail to activate the desired clusters. Genetic manipulation to overexpress pathway-specific regulators or delete repressors can be technically challenging and is not always feasible, particularly in genetically intractable strains. Moreover, existing screening techniques often lack the sensitivity or throughput required to systematically identify small molecule elicitors capable of derepressing these pathways. As a result, the vast majority of cryptic metabolites encoded in bacterial genomes remain inaccessible, hindering efforts to discover new bioactive compounds.

Technology

The High-Throughput Elicitor Screens (HiTES) platform is an advanced chemogenetic screening system designed to unlock the vast, untapped reservoir of natural products encoded by silent biosynthetic gene clusters (BGCs) in actinomycete bacteria, particularly Streptomyces species. This technology integrates fluorescent reporter gene constructs—such as multiple copies of eGFP—into the target BGC or a neutral chromosomal site, enabling sensitive, high-throughput detection of gene cluster activation in response to small molecule elicitors. By systematically exposing engineered reporter strains to diverse chemical libraries in microplate formats, HiTES identifies compounds that induce significant increases in fluorescence, indicating activation of otherwise silent BGCs. Subsequent validation steps include transcript analysis, dose-response assays, and comprehensive metabolite characterization using HPLC-Qtof-MS and NMR spectroscopy, leading to the discovery of novel bioactive compounds with antifungal, antibacterial, or protease-inhibiting properties.

What differentiates this technology is its ability to systematically and efficiently activate and study silent BGCs that are typically inaccessible under standard laboratory conditions. Unlike traditional methods that rely on random mutagenesis or labor-intensive genetic manipulation, HiTES leverages a tunable, high-throughput approach that combines chemical elicitation with sensitive, real-time genetic reporters. The platform not only pinpoints small molecules capable of derepressing specific gene clusters—often by downregulating pathway-specific repressors like SurR—but also enables rapid validation and structural elucidation of induced metabolites. Its modular design allows for broad applicability across various bacterial species and gene clusters, facilitating the scalable discovery of new natural products for drug development. This comprehensive workflow, from activation to bioactivity assessment, positions HiTES as a transformative tool in natural product discovery and synthetic biology.

Benefits

• Enables activation of silent biosynthetic gene clusters (BGCs) in actinomycete bacteria, unlocking novel natural products.
• Utilizes sensitive fluorescent reporter systems for high-throughput screening of small molecule elicitors.
• Facilitates discovery and structural characterization of new bioactive secondary metabolites with antifungal, antibacterial, and protease-inhibiting activities.
• Provides mechanistic insights by identifying transcriptional repressors and their modulation to derepress biosynthetic pathways.
• Combines genetic engineering and chemical elicitation for tunable and enhanced metabolite production.
• Applicable broadly to diverse bacterial species, accelerating natural product drug discovery and development.
• Supports downstream bioassays and semi-synthetic modification of newly discovered compounds for pharmaceutical applications.

Applications

• Antibiotic drug discovery
• Anticancer compound identification
• Antifungal agent development
• Protease inhibitor screening
• Natural product library expansion

Additional info

The HiTES platform activates silent biosynthetic gene clusters in actinomycete bacteria using fluorescent reporter constructs, such as eGFP, to monitor transcriptional activity. By screening small molecule libraries, specific elicitors are identified that trigger gene expression, often by downregulating pathway repressors like SurR. This chemogenetic approach enables the discovery and structural characterization of novel bioactive secondary metabolites, including cyclic octapeptides and quinones, through mass spectrometry and NMR spectroscopy.