Understanding and Exploiting Fungal Bisorbicillinoid Biosynthesis

索比西林类化合物是一类特殊的真菌代谢产物，具有广泛的结构和生物活性。在以前的工作中，我们建立了关键前体山梨西林醇的生物合成路线。然而，后期剪裁步骤，这是至关重要的生物多样性，尚未阐明。真菌里氏木霉（Trichoderma reesei）QMa 6是一种多产的索比西林类物质的生产者，并且基因组是可药用的。我们已经确定了一个基因簇的山梨西林类生物合成，并确认其功能的单加氧酶基因SorbC的靶向基因敲除（KO），这导致的损失山梨西林类产品。山梨西林醇类似物的二聚化反应是自发的还是酶催化的4+2环加成反应，是山梨西林生物合成的一个基本问题。我们已经建立了可靠的索比西林生产条件和有效的转化方法。reesei。我们还在T. reesei，我们现在希望开始研究生物合成基因簇中每个基因的功能，以确定整个生物合成途径和山梨西林醇的二聚化如何发生，以及获得生物合成中间体。此外，我们在使用宿主真菌Aspergillus sp.的真菌生物合成基因簇的异源表达方面具有广泛的专业知识。我们将从生物合成簇中单独克隆和表达每个基因，以确认整个生物合成途径并产生更多的生物合成中间体。同时，我们计划利用体外方法研究索比西林的生物合成，包括在大肠杆菌中异源表达索比西林生物合成基因。杆菌这将使我们能够研究由簇编码的不同酶的底物选择性，使用从基因KO和异源表达实验产生的天然底物，以及引入非天然底物。如果我们从我们的基因敲除和异源表达实验中鉴定出4+2环加成候选物，我们将拥有充分的专业知识来对这种独特的酶进行生物化学表征，并确认其功能和底物选择性。最后，通过与Hemholtz感染研究和PD中心的Marc Stadler教授合作。Leibniz Universität的Carsten Zeilinger博士表示，所有天然存在的山梨西林类化合物，基因KO /表达的途径中间体和体外酶试验产生的非天然化合物，将在广泛的生物试验中进行测试，包括但不限于抗癌和抗微生物活性。

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.