Discovery of Cryptic Natural Products Using High-Throughput Elicitor Screening on Agar Media

Professor Mo Seyedsayamdost

Professor Mohammad R. “Mo” Seyedsayamdost runs the Mo LAB in Princeton’s Dept. of Chemistry.  Ten years ago he developed a high-throughput screening approach referred to as HiTES (High-Throughput Elicitor Screening) using libraries of small molecule elicitors to activate silent/cryptic bacterial biosynthetic gene clusters which could greatly expand the repertoire of therapeutic natural products.  His lab continues to develop the HiTES approach on agar instead of liquid media to access the products of cryptic biosynthetic gene clusters in two Burkholderia species.  The new agar HiTES approach methodology and results are summarized as follows:

- High-throughput elicitor screening (HiTES) in a 96-well plate format with robotic addition of elicitor compounds

- UPLC-Qtof-MS analysis of the metabolites produced in the HiTES assay

- 3D metabolomic mapping using MetEx software to analyze the UPLC-Qtof-MS data

- Large-scale agar cultures with elicitor compounds to produce and purify novel metabolites

- 1D and 2D NMR spectroscopy to elucidate the structures of the purified metabolites

- They successfully applied an agar-based high-throughput elicitor screening (HiTES) approach to induce the production of five new cryptic natural products, including two unusual burkethyl compounds, in Burkholderia plantarii and Burkholderia gladioli, which were not observed in liquid cultures.

- They identified the biosynthetic gene cluster responsible for the production of the two new burkethyl compounds in B. plantarii. 

- They also discovered three new gladiobactin analogs in B. gladioli using the agar-based HiTES approach.

Agar-based HiTES is a promising approach for discovering new natural products, even from bacterial strains that have been extensively studied before.

HiTES on solid agar media. (a) Cells are grown in “mini-petri dishes” on agar in the presence of potential elicitors. The induced metabolomes are then extracted and subjected to UPLC-Qtof-MS to identify cryptic metabolites. (b, c) Application of agar-based HiTES to B. plantarii (b) and B. gladioli (c). Metabolites that are overproduced or induced are shown in 3D maps displaying the m/z and intensity for each compound as a function of elicitor.

Lee SR, Gallant É, Seyedsayamdost MR. Discovery of Cryptic Natural Products Using High-Throughput Elicitor Screening on Agar Media. Biochemistry. 2025 Jan 7;64(1):20-25. doi: 10.1021/acs.biochem.4c00659. Epub 2024 Dec 10. PMID: 39655417.  https://pubs.acs.org/doi/10.1021/acs.biochem.4c00659