Directed evolution of nitrogenase
File(s)
Author(s)
Bennett, Emily
Type
Thesis
Abstract
Nitrogen-fixation, which converts gaseous nitrogen (N2) into bioavailable ammonia (NH3) is a critically important reaction in the biosphere. Industrial nitrogen-fixation is an energy- intensive chemical reaction that produces NH3-based fertilisers. Fertiliser use dramatically increases crop yield but often causes environmental pollution. Biological nitrogen-fixation, catalysed by nitrogenase, offers an environmentally friendly alternative to meet the global nitrogen demand. Thus, synthetic biology aims to replace industrial nitrogen-fixation in agriculture with engineered strains expressing nitrogenase. This project aimed to evolve a minimal nitrogenase gene cluster from Paenibacillus polymyxa sp. WLY78 in E. coli to increase activity. A mathematical model describing RBS evolution was developed which links selection parameters to global mutation rate and library design for expression-strength optimisation. To enable high-throughput screening of nitrogenase library variants, I developed an anaerobic ammonia-sensitive fluorescent reporter. Modelling of ALE experiments revealed that balancing growth rate and passage size is essential. A selective nCas9-targeted system was developed which can diversify RBS sequences of an operon simultaneously or individually...
Version
Open Access
Date Issued
2023-08-08
Date Awarded
01/11/2023
License URL
Advisor
Isalan, Mark
Murray, James
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Grant Number
1949015
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)