Understanding and Exploiting Fungal Bisorbicillinoid Biosynthesis

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The sorbicillinoids are a priviledged family of fungal metabolites with a wide range of structures and biological activities. In previous work, we established the biosynthetic route to the key precursor sorbicillinol. However, the late tailoring steps, which are crucial for the biological diversity, have not yet been elucidated. The fungus Trichoderma reesei QMa6 is a prolific producer of sorbicillinoids and the genome is publically available. We have identified a gene cluster for sorbicillinoid biosynthesis, and confirmed its function by targeted gene knockout (KO) of the monooxygenase gene SorbC, which resulted in the loss of sorbicillinoid production. We now plan to investigate and exploit sorbicillin biosynthesis for the production of new bio-active compounds.A fundamental question in the biosynthesis of the sorbicillinoids is whether dimerization of sorbicillinol analogues is a spontaneous or enzyme-catalysed 4+2 cycloaddition reaction. We have developed reliable sorbicillinoid production conditions and an efficient transformation method for T. reesei. We have also demonstrated effective gene KO in T. reesei, and we now wish to start investigating the function of each gene in the biosynthetic gene cluster to determine the overall biosynthetic pathway and how dimerization of sorbicillinol occurs as well as access biosynthetic intermediates. In addition we have extensive expertise in the heterologous expression of fungal biosynthetic gene clusters using the host fungus Aspergillus oryzae. We will individually clone and express each gene from the biosynthetic cluster to confirm the overall biosynthetic pathway and generate more biosynthetic intermediates. In parallel, we plan to investigate sorbicillinoid biosynthesis using in-vitro methods including heterologous expression of sorbicillin biosynthetic genes in E. coli. This will enable us to investigate the substrate selectivity of different enzymes encoded by the cluster using natural substrates generated from gene KO and heterologous expression experiments, as well as introduce unnatural substrates. Should we identify a 4+2 cycloaddition candidate from our gene knockout and heterologous expression experiments, we have the expertise to fully biochemically characterize this unique enzyme and confirm its function and substrate selectivity. Finally, through collaborations with Professor Marc Stadler at the Hemholtz Centre for Infection research and PD. Dr. Carsten Zeilinger at Leibniz Universität, all naturally occurring sorbicillinoids, pathway intermediates from gene KO / expression and unnatural compounds generated from in-vitro enzyme assays, will be tested in a wide range of biological assays, including, but not limited to, anti-cancer and anti-microbial activities.