Directed Evolution Approaches to Manipulation of Biological Pathways

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

The motivation behind this proposal is best summarised in the words of Thomas Alva Edison: Until man reproduces a blade of grass Nature will laugh at our so-called scientific knowledge. This proposal is part of a long-term effort aimed at reproducing Nature s remarkable ability to generate molecular machines that perform at levels near perfection. Most biological processes involve the action of enzymes, which are central for explaining the workings of living cells and organisms. However, there is no comprehensive quantitative understanding of enzyme action and our current understanding certainly fails the most severe test ? that of producing catalysts with rates that rival natural enzymes. We want to address this complex question by evolution experiments in the test tube, harnessing the forces of Darwinian evolution. The evolutionary snapshots should then tell us how a catalytic machineries involved in signalling cascades and the biosyntheis of un-natural natural products (as potential drug candidates) are built up ? and generate useful reagents that may make it possible to probe signaling pathways and make new potential drug candidates.

Technical Summary

By mechanistic understanding of biological processes starting at tthe basic level of mechanistic organic chemistry in enzyme active sites, i plan to work towards a knowledge base fro understanding the molecular basis of diseases, to probe and manipulate cellular processes, to generate natural products with new and altered properties or to produce environmentally friendly, high-quality reagents for application in synthetic chemistry and biotechnology. To this end I use a new in vitro evolution technology (GENESCIS) that allows multiple rounds of selection from libraries of 10e10 memebers directly for catalysis. Evolution of new activities is based on mechanistic ratinoales and identified catalytic solutions will provide snapshots of the evolution of mechanism.
In a first sub-project I focus on the enzymology of phosphokinase catalysis. This type of reaction is crucial for signal transduction pathways that can lead e.g. to reprogramming of the cell cycle and ultimately to cancer. By in vitro evolution, I want to achieve altered specificity, gradual increases of activity (for mutants with broadened specificity), and activity switches to the related sulfokinase reaction (based on transition state similarity and the observation of catalytic promiscuity. These mutant enzymes could then be tested for their physiological roles e.g. in signal transduction and provide tools to pin-point the roles of sulfate and phosphate transfer enzymes in the cellular machinery. In a second subproject (2.2) I plan to use in vitro evolution to understand and engineer multifunctional enzymes that operate as molecular assembly lines in non-ribosomal peptide biosynthesis, in particular gramicidin synthetase. Our general objective is to expand the scope of such natural system of combinatorial chemistry by directed evolution. Many peptide-based natural products of therapeutic importance are synthesised by nonribosomal peptide synthetases (NRPSs), such as the immunosuppressants cyclosporine, the ?antibiotic of last resort? vancomycin and part-structures of the anti-cancer agent epothilone or the beta-lactam antibiotics. Our proposal aims at mixing and matching enzyme modules to allow the generation of novel compounds - natural products and new, non-natural analogs that are inaccessible straightforwardly by synthetic chemistry. We believe that altering and swapping modules could give rise to novel protein templates that can be used to assemble and produce expanded repertoires of natural-product-like compounds of rich therapeutical potential.

Publications

10 25 50

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Van Loo B (2010) An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily. in Proceedings of the National Academy of Sciences of the United States of America

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Villiers BR (2009) Mapping the limits of substrate specificity of the adenylation domain of TycA. in Chembiochem : a European journal of chemical biology

 
Description EU Marie-Curie ITN:European Network on Evolution of FUnctional Proteins
Amount £2,200,000 (GBP)
Funding ID 215560 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start  
 
Description Sulfatases 
Organisation French National Institute of Agricultural Research
Department Department of Biochemistry
Country France 
Sector Public 
PI Contribution Protein expression, structural and functional characterisation and kinetic analysis (95%).
Collaborator Contribution Knowledge about modification of active site Cys to fGly.
Impact See our PNAS paper (van Loo et al, 2010) in list of publications.
Start Year 2007
 
Description BBC Radio The Naked Scientist 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Radio Interview on my research activities.

Audience feedback.
Year(s) Of Engagement Activity 2008
 
Description ELRIG Drug Discovery Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact Presentation to industrial audience.

Industrial funding and collaboration.
Year(s) Of Engagement Activity 2008