This project delivers a better understanding of how Polyketide synthases (PKS) build an assembly line for production of complex secondary metabolites by transferring intermediates between different enzymatic sites. The insights gained here are an important prerequisite for successful engineering of PKS to obtain new products.
In this study, we analyzed molecular mechanism of substrate transfer in fungal non-reducing PKS (NR-PKS). Structural analysis of trapped transient states by cryo-electron microscopy (cryoEM) provides the first atomistic insights into transfer of intermediates in PKS assembly lines. So far, we have achieved high resolution analysis of two out of the possible three states of substrate transfer required for chain initiation and elongation, providing a full visualization of the interaction of the mobile, substrate-loaded acyl carrier protein (ACP) at two enzymatic sites, the ketosynthase and the starter-acyl transferase domain. These results are a major breakthrough for understanding substrate transfer in PKS already, but we aim to complete the visualization also for the third step, occurring at the malonyl-acyl transferase, for which we currently only have intermediate resolution data. Based on our structural analysis in the AL4Bioch project we have designed and produced variant proteins for analyzing effects of modifications in the carrier protein binding interfaces on substrate transfer in PKS. Key findings of our work include the revelation of the different modes of carrier-protein recognition and of effects of carrier protein interaction on the overall protein assembly.
An important aspect of this project is to publish results in a scientific journal of highest-level. To achieve such a level scientific publication, we aim for as complete representation of substrate transfer states in the chosen system as possible. Unfortunately, the AL4Bioch was massively and directly affected by the SARS-Cov2 crisis. Because my daughter lives in Spain, I experienced a series of family-related difficulties. In the beginning of the pandemic, Spain was heavily affected by a 1st Coronavirus wave with a hard lockdown and full of restrictions. I spent two months taking care of my daughter because her mother was recruited as nurse at Hospital to fight the pandemic. During that period, I was the main figure for my daughter. Kindly, the University of Basel decided to support an extension of AL4Bioch project and paid two additional months of helping me to advance in my H2020 project. This allowed me to focus on aim 4 of the proposal (Functional validation and PKS engineering). Beyond these two months core period, lab work and scientific interactions were still affected during most of the project duration. The established collaboration with the University of Baltimore was also affected by COVID-19 situation. Under normal circumstances, I would have travelled to Baltimore (US) to interacts with our collaborators on the chemistry part of this project and to carry out biochemical experiments. Under the unfortunate circumstances, we have obtained the strongest possible results, but still requires several extra time to achieve our highest aims as a prerequisite to properly disseminate the work as planned. Unfortunately, the Covid pandemic have prevented many of the planned in-person dissemination activities and interactions with the public. Still, our scientific results could be shared in seven occasions at scientific meetings and workshops, but latest advances and publication have not submitted yet.
