Biosynthetic studies on tropolone maleic acid anhydride conjugates from Hypoxylon lienhwacheense Katharina Schmidt, Jin Feng, Eric Kuhnert, Russell J. Cox Leibniz Universität Hannover, Germany The fungus Hypoxylon lienhwachseense is a producer of tropolone maleic acid anhydride conjugates, called lienhwalides. Two PKS-containing biosynthetic gene clusters (BGC) with high homology to genes from well-known biosynthetic pathways were found by genome analysis. The biosynthesis of the tropolone compounds in lienhwalides was investigated based on related compounds (e.g. stipitatic acid biosynthesis in T. stipitatus ). Heterologous expression experiments with Aspergillus oryzae as a host elucidated the first steps of the biosynthesis to form the intermediate early products. Further modifications in the pathway, which lead to the tropolone antibiotic C, are studied by enzymatic in vitro experiments. On the other hand, the BGC responsible for the maleic acid anhydride moiety was analysed based on other maleidride producers (e.g. byssochlamic acid from B. fulva ). Heterologous expression experiments proved the production of maleic acid anhydride monomers by a set of five core genes. Interestingly, another natural product called cordyanhydride B can be found during cultivation of the wild type fungus. Cordyanhydride B consists of three maleic acid anhydride monomers, linked together linearly. The investigation of the linkage of the maleic acid anhydride units are still ongoing. Further aspects of the biosynthesis are still unknown and will be the focus of future work, including studies on later tailoring reactions in the biosynthetic pathways as well as the linking of the tropolone and maleic acid anhydride compounds for the formation of the final lienhwalides. References 1. Davison, J., al Fahad, A., Cai, M., Song, Z., Yehia, S. Y., Lazarus, C. M., Bailey, A. M., Simpson, T. J., and Cox, R. J. (2012) Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis, Proceedings of the National Academy of Sciences of the United States of America 109, 7642–7647 published online Apr 16, 2012. DOI: 10.1073/pnas.1201469109. 2. Gerke, J., Köhler, A. M., Wennrich, J.-P., Große, V., Shao, L., Heinrich, A. K., Bode, H. B., Chen, W., Surup, F., and Braus, G. H. (2022) Biosynthesis of Antibacterial Iron-Chelating Tropolones in Aspergillus nidulans as Response to Glycopeptide- Producing Streptomycetes, Front. Fungal Biol. 2. DOI: 10.3389/ffunb.2021.777474. 3. Kuhnert, E., Navarro-Muñoz, J. C., Becker, K., Stadler, M., Collemare, J., and Cox, R. J. (2021) Secondary metabolite biosynthetic diversity in the fungal family Hypoxylaceae and Xylaria hypoxylon, Studies in mycology 99, 100118 published online Aug 26, 2021. DOI: 10.1016/j.simyco.2021.100118. 4. Schotte, C., Li, L., Wibberg, D., Kalinowski, J., and Cox, R. J. (2020) Synthetic Biology Driven Biosynthesis of Unnatural Tropolone Sesquiterpenoids, Angewandte Chemie (International ed. in English) 59, 23870–23878 published online Oct 26, 2020. DOI: 10.1002/anie.202009914. 5. Williams, K., Szwalbe, A. J., Mulholland, N. P., Vincent, J. L., Bailey, A. M., Willis, C. L., Simpson, T. J., and Cox, R. J. (2016) Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis, Angewandte Chemie (International ed. in English) 55, 6784–6788 published online Apr 21, 2016. DOI: 10.1002/anie.201511882.
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