The adaptive laboratory evolution of an Escherichia coli strain with increased tolerance to styrene and ethylbenzene
Publication Date
August 4, 2021
Creator
Abstract
Microbial bioprocesses could provide sustainable alternatives to many of the current manufacturing processes in the chemical industry. A significant amount of research and development interest is currently invested in bio-based production routes to petroleum derived aromatic monomers. However, the bio-based production of these aromatic monomers is not on a commercial scale due to product toxicity which inhibits production titres. Styrene is a mono-aromatic organic compound commonly used as an aromatic monomer in the production of many synthetic rubbers, co-polymers and polystyrene plastics. The biosynthesis of styrene has been achieved from a glucose feedstock in Escherichia coli via the construction of a novel pathway. The achievable bio-production titres for styrene in an E. coli production strain, is close to the toxicity threshold concentration, which is reported at 2.9 mmol L-1. Increasing the inherent tolerance of the production strain is an ideal strategy to increase styrene production titres. However, the rational targeting of specific genes/gene product may not reveal all the factors, such as multiple mutations, that underpins the tolerance phenotype. Adaptive laboratory evolution (ALE) is a method used to generate adaptive beneficial mutations in a population, by propagating a microbial population under a selection pressure and subsequently screening to isolate the adaptive mutant. ALE was explored as a method to generate E. coli BW25113 strains with a tolerance to a final styrene concentration above the toxicity threshold of 2.9 mmol L-1. Extensive method development was carried out to allow analytical control over the concentration of styrene, supplied as the selection pressure to a population of E. coli BW25113 cells. This provided useful insight into conducting ALE with other chemicals with similar physicochemical properties to styrene. The ALE experiments carried out, uncovered some new insights into the toxicity mechanisms of styrene not only being centred on its hydrophobicity and ease of entering cell membranes; but also due to the double bond located on its aromatic ring substituent. A novel two stage ALE strategy was implemented to adapt the E. coli BW25113 strain; firstly, to evolve resistance to the toxicity effects relating to the hydrophobicity of the aromatic ring and then to the reactive double bond of styrene.
This was done by using ethylbenzene, a structurally related compound that lacks the double bond, as a selective pressure prior to further screening using styrene. This ALE method generated an E. coli BW25113 styrene tolerant mutant which could grow in 4 mmol L-1 styrene concentration. Subsequent genome resequencing of the mutant revealed mutations in gene commonly associated with chemical tolerance in novel locations. Findings gained from this study provide strategies to conduct ALE to evolve microbial tolerance to other hydrophobic compounds and potential gene targets which can be rationally engineered.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
Department of Chemical and Environmental Engineering (UK)
eprints ID
64721
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