Developing a Biomanufacturing Platform for the Site-Selective Functionalization and Structural Diversification of Cytochalasan-Based Carbon Skeletons

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Fungi can synthesize small molecules with complex structures using a number of highly coordinated enzymes. These molecules are difficult to make synthetically, and they can aid in crop production or have beneficial human health effects. This project will identify fungal enzymes that efficiently modify more than one substrate in a predictable way. Also, enzymes will be engineered to expand the range of substrates. A biomanufacturing platform to synthesize bioactive molecules at lower costs will be the end result. In this project, students will be trained in core STEM technologies, with a focus on commercial applications.Cytochalasans are phytotoxic, cytotoxic and actin-binding natural products. Produced by fungi, over 400 variants have been described. The structural diversity is partly explained by the flexibility of the enzymes that introduce and modify functional groups. These enzymes structurally rearrange the core carbon skeleton in a site-selective manner, often on more than one substrate. Genome mining will be used to identify cytochalasan tailoring enzymes. Overproducing strains will be characterized chemically. Transcription factor over-expression will be investigated. Targeted gene knock-out will confirm the function and scope of the enzymes. The enzymes will be engineered to expand their substrate range. Synthetic biology and metabolic engineering tools will be used to generate a biomanufacturing platform for large scale production and functionalization of cytochalasan-based carbon skeletons. This project will generate fundamental knowledge on how to effectively engineer enzymes and metabolic pathways for efficient and rapid structural diversification of bioactive molecules with commercial potential.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.