Genetics independent readout for high throughput elicitor screens using flow injection DAD-MS Ignacio Pérez-Victoria, Denise M. Selegato, Marta González, Jesús Martín, Sandra Resa, Daniel Oves-Costales, Olga Genilloud and Fernando Reyes Fundación MEDINA, Spain The high-throughput elicitor screens (HiTES) approach has been developed as a strategy to trigger the expression of otherwise “silent” biosynthetic gene clusters (BGCs) in microorganisms 1 . In its original implementation, a reporter gene was inserted into the silent BGC of interest to provide a fast fluorescence- based readout for its expression when such reporter strain mutant was screened against small molecule libraries to identify candidate elicitors [1]. A limitation of the approach was the requirement for genetic manipulation of the studied strains, what made it time consuming and non-suitable for unsequenced or genetically intractable microorganisms. For this reason, alternative genetic-free readouts for HiTES have been evaluated, such as those based on colorimetry 2 or bioactivity 3 . The most universal HiTES readouts implemented so far though are based on mass spectrometry (MS) detection. For example, laser ablation electrospray ionization (LAESI) MS was employed to rapidly survey the induced metabolomes by some elicitors to identify new cryptic metabolites 4 . Likewise, matrix assisted laser desorption/ionization (MALDI) MS has been coupled to HiTES with special interest in the detection of cryptic nonribosomal peptides 5 . More recently, UPLC-MS has been employed as HiTES readout taking advantage of the extra retention time dimension to avoid possible issues related to ion suppression in complex extracts 6 . To extend the universality of MS-based readout for HiTES we propose herein the use of flow injection analysis (FIA) with ESI-MS detection, FIE-MS, carried out on typical low-resolution HPLC-MS equipment, employing the LC instrument as the actual flow injection module to introduce the sample in the running solvent as a ‘plug flow’. FIE-MS is a robust technique for high-throughput non-targeted global metabolome fingerprinting 7 being thus a very appropriate readout for evaluating the induction of SMs in HiTES experiments. In fact, related direct infusion ESI-MS had already been employed for the rapid assessment of SM presence in actinomycetes 8 . To further enhance the approach, taking advantage of the integrated DAD detector in the HPLC instrument, we propose to register additionally the UV-vis absorbance of all HiTES derived samples to facilitate tracking the induction of colored compounds difficult to observe visually as a phenotypic feature due to the interference from the fermentation media background. The DAD and MS spectra are acquired sequentially leading to a flow injection DAD-MS analytical scheme which can be considered as an LC-DAD-MS analysis without chromatographic column. The validity and usefulness of this approach as a readout for HiTES is demonstrated herein using the model actinomycete Streptomyces albus J1074 and a custom library of 560 organic compounds. References
1. M.R. Seyedsayamdost, PNAS 2014 , 111(20), 7266-7271. 2. A. Craney et al., Chem. Biol. 2012 , 19(8), 1020-1027. 3. K. Moon et al., ACS Chem. Biol. 2019 , 14(4), 767-774. 4. F. Xu et al., Nature Chem. Biol. 2019 , 15(2), 161-168.
5. C. Zhang, M.R. Seyedsayamdost, Angew. Chem. Int. Ed. 2020 , 59(51),23005-23009. 6. B. C. Covington, M. R. Seyedsayamdost, ACS Chem. Biol. 2021 , 16(12), 2825-2833. 7. J. Draper et al., Metabolomics 2013 , 9(1), 4-29. 8. R.E. Higgs et al., Appl. Environ. Microbiol. 2001 , 67(1), 371-376.
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