Engineering Cytochalasan Biosynthesis for the Production of New Molecular Tools

• Applicant: Professor Dr. Russell J. Cox
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 567649639

Cytochalasans are fungal metabolites that are potent ligands of eukaryotic actin and important tools for the investigation of actin biochemistry in vitro and for cell-biology studies of cellular motility and metastasis in vivo. However, the availability of cytochalasans is limited, and this has impeded progress in these areas. During the first phase of this project an effective total biosynthesis platform for the production of cytochalasans was established in Aspergillus nidulans. This allows the synthesis of cytochalasans by the assembly of biosynthetic genes. New cytochalasans can be produced much more rapidly than by synthetic chemistry, but with more limited chemical diversity. This project aims to convert the production platform into an engineering platform and test the limits of structural engineering. The project has two main goals. First, we aim to vary the amino-acid derived portion of the cytochalasan skeleton by systematically investigating and engineering the adenylation (A) domain of the nonribosomal peptide synthetase (NRPS) module of the cytochalasan synthetase. Fungal A-domains have received much less attention than the corresponding catalysts in bacteria, and their selectivities are only understood in outline detail. We will systematically vary and engineer the A-domains to obtain new cytochalasan skeletons and provide new information about their selectivities. New compounds produced will be tested for biological activity in vitro and in vivo by collaboration partners. We will also study the A-domain selectivity in vitro using substrates and assays already available. Second, we aim to understand the key Knoevenagel and Diels-Alder ring-forming biochemical processes that build the distinctive hexahydroisoindolone core of the cytochalasans. While the chemistry of these steps appears simple on paper, key biochemical questions remain about how they are controlled to avoid thermochemical shunt pathways observed in the absence of protein catalysts. Here, we will work in vitro to understand the biochemistry of the PyiE/PyiF proteins. We will also collaborate with structural biologists in Hannover (Köhnke group) to obtain structural details of the ring-forming enzymes and fungal A-domains. Overall we will: generate new knowledge about the selectivity of fungal A-domains; generate new methods for the rapid total biosynthesis and engineering of natural products; produce new cytochalasans with high potential as tools to study the in vitro and in vivo activity of actin; and discover new information about the biochemical steps involved in the back-to-back Knoevenagel and Diels-Alder cyclisations during cytochalasan biosynthesis.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Jennifer Gerke