Studies on pleurotin-producing fungi and heterologous hosts for elucidation of biosynthesis Jack Weaver 1 , Duha Alkhder 1 , Dr Emzo De Los Santos 3 , Dr Lijiang Song 2 , Dr Christophe Corre 1,2 and Dr Fabrizio Alberti 1 1 School of Life Sciences, University of Warwick, UK, 2 Department of Chemistry, University of Warwick, UK, 3 UCB, UK Pleurotin, a bioactive meroterpenoid, made by Hohenbuehelia atrocaerulea , has antibiotic (1), anticancer properties (2), and a congener inhibits the hepatitis C virus (3). Long fermentation times for H. atrocaerulea , and the inability to manufacture stereo-selectively (4), have kept it neglected in the pharmaceutical industry. Previous research has identified proven intermediates (5) and a predicted metabolic pathway was produced. Modern biotechnological and chemical techniques now make it possible to recreate the biosynthetic pathway within heterologous organisms. For this purpose we selected Saccharomyces cerevisiae and Aspergillus oryzae , due to their fast growth times, easy growth requirements, and for being well understood organisms utilised across both academia and industry. Using short-read and long-read DNA sequencing, combined with RNA sequencing we have assembled and annotated a robust genome. This genome was the foundation on which transcriptomic analyses was performed. Leading to the identification of a putative biosynthetic gene cluster significantly upregulated during pleurotin production. More evidence is also being collected using proteomic techniques, to identify any further proteins potentially associated with pleurotin biosynthesis. We are expressing putative pathway genes heterologously to discover intermediates and characterise the entire pathway, while simultaneously developing a base strain to optimise for pleurotin production. Purified metabolites from these strains are being analysed through NMR spectroscopy and have so far confirmed the presence of known precursors. References 1. Robbins, W., Kavanagh, F. and Hervey, A., 1947. Antibiotic Substances from Basidiomycetes: I. Pleurotus griseus. Proceedings of the National Academy of Sciences, 33(6), pp.171-176. 2. Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G, Powis G. Antitumor activity and pharmacodynamic properties of PX- 478, an inhibitor of hypoxia-inducible factor-1alpha. Mol Cancer Ther. 2004 Mar;3(3):233-44. 3. Sandargo, B., Thongbai, B., Praditya, D., Steinmann, E., Stadler, M. and Surup, F., 2018. Antiviral 4- Hydroxypleurogrisein and Antimicrobial Pleurotin Derivatives from Cultures of the Nematophagous Basidiomycete Hohenbuehelia grisea. Molecules, 23(10), p.2697. 4. Hart, D. and Huang, H., 1988. Total synthesis of (.+-.)-pleurotin and (.+-.)-dihydropleurotin acid. Journal of the American Chemical Society, 110(5), pp.1634-1635.5. Vogt, Peter. 1982. Zur Biosynthese vo Pleurotin. Doctoral Thesis, ETH Zürich.
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