Understanding and engineering the factors that control fatty acid biosynthesis in escherichia coli
File(s)
Author(s)
Gallagher, Danielle
Type
Thesis
Abstract
This research identified and characterised several genetic and environmental factors that contribute to controlling the metabolic flux through fatty acid synthesis (FAS) in Escherichia coli, to enable high yield in production of fatty acids (FA) which can be further processed towards industrially relevant commodity products. Due to their essentiality to growth, high energy and carbon investment, E. coli have evolved
several mechanisms that tightly coordinate FAS with phospholipid synthesis and the energy status of the cell. This makes the outcome of systematic engineering manipulations difficult to predict, but is an area of research that benefits greatly from
computationally driven solutions that can be gained from modelling metabolism. In order to model metabolism effectively, parameters of the kinetic system must be obtained and enable subsequent associations with in vivo metabolism and physiology. To adequately make this association, mathematical formulas describing biochemical pathways must account for the control and regulation the system is
subject to, so that metabolic engineers can design towards specific phenotypes and product yield.
This work investigated FAS regulation under a range of perturbations and how the system responds in vivo to these changes, to gain insight into the control and
regulations that are in place during fluctuating conditions. Furthermore, the experiments of this study found adaptive evolution to FA to be a promising strategy in complementing a directed engineering approach in E. coli, in order to adapt to the burden that presents itself during FA overproduction. As a directed approach towards FA overproduction, three novel bypass routes to malonyl-CoA production were also investigated, and found to improve in vivo rates of malonyl-CoA. However,
improving this rate-limiting step alone was not sufficient for to overcome the native regulatory and energetic limitations that are present in FAS. The bypass routes were therefore combined with specific process optimisations identified during this research, which was found to improve yield compared to non-optimised cultivations.
several mechanisms that tightly coordinate FAS with phospholipid synthesis and the energy status of the cell. This makes the outcome of systematic engineering manipulations difficult to predict, but is an area of research that benefits greatly from
computationally driven solutions that can be gained from modelling metabolism. In order to model metabolism effectively, parameters of the kinetic system must be obtained and enable subsequent associations with in vivo metabolism and physiology. To adequately make this association, mathematical formulas describing biochemical pathways must account for the control and regulation the system is
subject to, so that metabolic engineers can design towards specific phenotypes and product yield.
This work investigated FAS regulation under a range of perturbations and how the system responds in vivo to these changes, to gain insight into the control and
regulations that are in place during fluctuating conditions. Furthermore, the experiments of this study found adaptive evolution to FA to be a promising strategy in complementing a directed engineering approach in E. coli, in order to adapt to the burden that presents itself during FA overproduction. As a directed approach towards FA overproduction, three novel bypass routes to malonyl-CoA production were also investigated, and found to improve in vivo rates of malonyl-CoA. However,
improving this rate-limiting step alone was not sufficient for to overcome the native regulatory and energetic limitations that are present in FAS. The bypass routes were therefore combined with specific process optimisations identified during this research, which was found to improve yield compared to non-optimised cultivations.
Version
Open Access
Date Issued
2018-09
Date Awarded
2019-02
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
License URL
Advisor
Jones, Patrik
Sponsor
European Union
Grant Number
P51198
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)