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Menchavez, Russel (2020) Host cell engineering for the production of methacrylate esters. PhD thesis, University of Nottingham.

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Abstract

Microbial bioprocess serves as an alternative route for the sustainable production of a variety of chemicals. Recent bioprocess development efforts has allowed its application for the commercial production of certain industrially relevant chemicals. However, most are still in the exploratory or precommercialization stage due to a variety of bottlenecks that needs to be addressed prior to commercialization. This includes the bioprocess route being developed by Lucite International for the production of butyl methacrylate, which could be part of an integrated process for the production of methacrylate esters. In this bioprocess, commercial viability is attainable with a butyl methacrylate titre of 10-20% v/v. One of the bottlenecks in this proposed bioprocess is the toxicity of the bioproduct towards the production strain, which could limit the attainable product titre. A previous study on its toxicity led to the isolation of E. coli strains that can grow vial cultures with BMA at 20% v/v. However, these strains were unable to demonstrate tolerance in a well-mixed environment. Thus, there is still a need to develop a robust host strain that can tolerate butyl methacrylate at the desired product titre.

E. coli BW25113 was explored as the potential host strain. Adaptive evolution via sequential batch and chemostat cultures were used to generate E. coli strains with tolerance for butyl methacrylate at 20% v/v. Genome shuffling was also used to further improve growth of E. coli with butyl methacrylate at 20% v/v. The possible mechanisms of tolerance for butyl methacrylate were determined with the use of genomic DNA and RNA sequencing of the evolved strains. The ability of the evolved strains to produce BMA was also tested by introduction of the heterologous pathway.

Adaptive evolution, through sequential batch and chemostat cultures, was successful in generating various E. coli strains with improved growth in the presence of BMA up to 20% v/v. Each of the evolved strains acquired various mutations that include an acrR mutation along with either a marR, soxR, and rob. The mutations acquired allowed increased expression in acrAB, which suggests that the AcrAB-TolC efflux pump might play an important part in the tolerance for butyl methacrylate. Exposure of the evolved strain to butyl methacrylate stimulated the activation of genes that belong to the oxidative stress, heat shock, phage shock, and acid stress response systems and membrane modifying, energy generating, and essential building block synthesizing enzymes. It also resulted in the repression of the genes related to DNA replication and protein synthesis. The use of the evolved strains as host cell for production did not show an improvement in butyl methacrylate titre in comparison to the parental strain. However, butyl methacrylate production seems to be limited by factors other than toxicity. Thus, there is a need for further investigation and improvement of the production pathway.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Stephens, Gill Conradie, Alex Pordea, Anca
Keywords:	sustainable bioprocess, host cell engineering, product tolerance, whole cell biocatalysis
Subjects:	T Technology > TP Chemical technology
Faculties/Schools:	UK Campuses > Faculty of Engineering
Item ID:	60034
Depositing User:	Menchavez, Russel
Date Deposited:	31 Jan 2023 09:17
Last Modified:	31 Jan 2023 09:19
URI:	https://eprints.nottingham.ac.uk/id/eprint/60034

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