Periodic Reporting for period 1 - AL4BIOCH (Assembly Lines for Biocombinatorial Chemistry)
Periodo di rendicontazione: 2019-05-01 al 2021-04-30
• What is the problem/issue being addressed?
The goal of this project is to study the functional architecture of Polyketide Synthetases (PKS) and the basis for their multidomain organization and to analyse intra- and intermolecular crosstalk in substrate transfer. We aim for a fundamental understanding of the structural organization of PKS biosynthesis, which provides a basis for PKS re-engineering in drug discovery and combinatorial biosynthesis.
• Why is it important for society?
Because PKS and their polyketide products are of outstanding relevance for fighting disease, this project in itself holds direct translational value, and a fundamental understanding of polyketide biosynthesis is crucial for extended targeted therapeutic intervention e.g. to overcome antibiotic resistances. The combined fundamental nature and high translational potential of this project suppose strongest impact and corresponding opportunities to publish with high impact.
• What are the overall objectives?
The main goal of this project is to contribute to fundamental understanding of molecular mechanisms in biology and to advance the fields of chemical biology and synthetic chemistry, biochemistry and natural product research, structural biology, as well as microbiology
The goal of this project is to study the functional architecture of Polyketide Synthetases (PKS) and the basis for their multidomain organization and to analyse intra- and intermolecular crosstalk in substrate transfer. We aim for a fundamental understanding of the structural organization of PKS biosynthesis, which provides a basis for PKS re-engineering in drug discovery and combinatorial biosynthesis.
• Why is it important for society?
Because PKS and their polyketide products are of outstanding relevance for fighting disease, this project in itself holds direct translational value, and a fundamental understanding of polyketide biosynthesis is crucial for extended targeted therapeutic intervention e.g. to overcome antibiotic resistances. The combined fundamental nature and high translational potential of this project suppose strongest impact and corresponding opportunities to publish with high impact.
• What are the overall objectives?
The main goal of this project is to contribute to fundamental understanding of molecular mechanisms in biology and to advance the fields of chemical biology and synthetic chemistry, biochemistry and natural product research, structural biology, as well as microbiology
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.
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.
The research was performed on modern and rapidly evolving techniques, including cryo electron microscopy (cryoEM), advanced computational image processing, biophysics, as well as chemical probes, which are most relevant for front-line molecular biology research.
In particular, AL4Bioch has obtained outstanding cryoEM reconstructions that visualize multi-domain polyketide synthetases reaction at high resolution, For instance, the full-length PksA protein structure reconstruction has an overall resolution of 2.7 A based on gold standard methods, something uncommon for such a dynamic molecular machine. This project provides snapshots of transient states in substrate loading and elongation of a PKS assembly line. These data are a key to understanding PKS assembly line biosynthesis and to design bio-inspired systems for green chemistry and a more sustainable future.
Apart from the high impact for society to obtain new molecules, a personal impact is to qualify for an early career independent research position in Europe. Excellent results for publication have been obtained, and are expected to lead to submission of a high-impact manuscript within several months. Establish a scientific network and international collaborations for independent projects has been a challenge during the Covid pandemics
In particular, AL4Bioch has obtained outstanding cryoEM reconstructions that visualize multi-domain polyketide synthetases reaction at high resolution, For instance, the full-length PksA protein structure reconstruction has an overall resolution of 2.7 A based on gold standard methods, something uncommon for such a dynamic molecular machine. This project provides snapshots of transient states in substrate loading and elongation of a PKS assembly line. These data are a key to understanding PKS assembly line biosynthesis and to design bio-inspired systems for green chemistry and a more sustainable future.
Apart from the high impact for society to obtain new molecules, a personal impact is to qualify for an early career independent research position in Europe. Excellent results for publication have been obtained, and are expected to lead to submission of a high-impact manuscript within several months. Establish a scientific network and international collaborations for independent projects has been a challenge during the Covid pandemics