Review of high throughput genetics free approach for activating secondary metabolite production in microbial species

Secondary metabolites constitute an important source of drug compounds for various diseases such as cancer and infectious diseases. However, recent efforts at uncovering novel secondary metabolites (natural products) have faced a significant roadblock in inability to activate expression of silent biosynthetic gene clusters (BGC). Specifically, many genes encoding enzymes partaking in the biosynthesis of secondary metabolites are either not expressed or expressed at a very low level under standard laboratory growth conditions. While approaches such as co-culture, manipulations of nutritional and growth conditions and genetic manipulations exist to aid in the activation of silent BGC, they are nevertheless time-consuming and low throughput. In Nature Chemical Biology, Xu and coworkers presents a genetics-free high throughput elicitor screening approach for activating silent BGC in different microbial species. Specifically, microbial species are cultivated in 96 well plates to which they are exposed to a library of 502 elicitors (inducers) able to activate the secondary metabolome of cells. Visualization of the secondary metabolites produced is aided by imaging mass spectrometry that affords fast, high throughput profiling of the secondary metabolome of cells with minimal sample preparation. Subsequently, putative inducers are validated by large scale cultivation experiments where structure elucidation of novel secondary metabolites is performed together with activity assays examining the antimicrobial properties of the new compounds. Compared to existing approaches, the authors’ method could activate multiple silent BGC simultaneously; thereby, enabling a greater diversity of secondary metabolites to be produced given possible cross-interaction effects between different BGCs. In addition, laser ablation electrospray ionization imaging mass spectrometry (LAESI-IMS) provides a high throughput tool for profiling the secondary metabolome of cells after induction in comparison to the long analysis time common in high performance liquid chromatography mass spectrometry (HPLC-MS). Validation of LAESI-IMS utility in the work expands the analytical toolkit of the field. However, the work is limited by the use of uniform growth and environmental conditions for testing the utility of different elicitors, where some of the elicitors may not be able to function under specific growth conditions. This might have limited the diversity of secondary metabolites produced. Additionally, ion suppression effects common in LAESI-IMS might have also precluded the detection of some secondary metabolites; thereby, restricting access to the full secondary metabolome of cells induced by different elicitors. Collectively, a high throughput genetics-free approach for activating silent BGC and visualizing the secondary metabolome induced has been validated through both 96 well plate assays and large-scale cultivation in shake flasks. Structure elucidation work reports the observation of novel secondary metabolites with unique molecular scaffolds that exhibit antimicrobial activity in inhibitory assays. The work, however, is limited by the use of common growth conditions for all inducers as well as ion suppression effect in IMS that restricts the set of secondary metabolites probed. Overall, the work expands the analytical toolkit of natural product research and would be a useful approach for high throughput drug discovery from microbial sources.