Study of the mechanism of action and biosynthesis of microbial secondary metabolites Duha Alkhder 1 , Fabrizio Alberti 2 , Nick Waterfield 1 , Irene Stefanini 1,3 1 The University of Warwick, UK, 2 School of Life Sciences and Department of Chemistry, University of Warwick, UK, 3 University of Turin, Italy 3,5-Dihydroxy-4-isopropyl-trans-stilbene (IPS) is a stilbene natural product made by Photorhabdus spp ., which are bacteria associated with entomopathogenic Heterorhabditis spp. nematodes. IPS has diverse biological and pharmacological properties, including antibacterial, antifungal, insecticidal, nematocidal, antioxidant and anticancer activities 1-3 . IPS has recently been approved as a treatment for psoriasis in China and Japan (under the commercial names of Benvitimod and Tapinarof, respectively). However, its mechanism of action remains unknown. The aim of this work is to investigate the mechanism of action of IPS in yeast and filamentous fungi. Previous analyses carried out by our group the group using a heterozygous S. cerevisiae deletion collection, identified 19 genes as being involved in pathways that may be potential targets of IPS. Interestingly, 7 of these identified genes are essential for the yeast survival, making them promising antifungal target candidates. To confirm the effect of IPS on products of these gene candidates, heterozygous recombinant strains were constructed by deleting the target genes through the integration of a KanMX cassette (conferring resistance to geneticin) in place of one of the two alleles. We found that yeast dis3 / dis3::KanMX mutant strain is more sensitive to IPS compared to the wild type S. cerevisiae W303 and mutant strains for the other potential targets. This suggests that DIS3 – responsible for RNA processing and degradation in both the nucleus and the cytoplasm - is a target of IPS. In order to study the mechanism of action of IPS in filamentous fungi, we overexpressed in Aspergillus oryzae the native ortholog of the yeast DIS3 gene, given that the deletion of a potentially essential gene in this haploid strain may have been lethal. The analysis of the mutant strain is currently underway. High-throughput molecular analyses, e.g. proteomic analysis and RNAseq is used to identify the exact pathways affected by IPS in both yeast and filamentous fungi.We observed that the yeast had an obvious response to IPS exposure, this was reflected in changes in the transcriptomic and proteomic levels of genes relating to many fundamental cellular processes. Our data is being compared with the work done in the Waterfield lab on the mode of action of IPS in Staphylococcus aureus 4 We will determine if IPS has the same or different targets in the eukaryote and
prokaryote. References 1. Li J. et al. (1995) Appl Environ Microbiol 61: 4329-4333. 2. Shi D. et al. (2017) J Agric Food Chem 65: 60-65. 3. Kumar S.N. et al. (2014) Ann Microbiol 64: 209-218. 4. Hapeshi A. et al . (2019) Microbiology 165: 516-526.
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