Thesis
Unravelling the stress responses and tolerance mechanisms of Pseudomonas putida KT2440 to methacrylate esters and monoaromatics
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2022
- Thesis identifier
- T16645
- Person Identifier (Local)
- 201869924
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The plastics industry is reliant on petrochemical feedstocks for the synthesis of large-volume commodity chemicals. The synthetic polymers poly(methyl methacrylate) (PMMA) and polystyrene (PS) are produced from petrochemically derived methyl methacrylate (MMA) and styrene respectively. In Europe and the US, MMA is largely produced through the acetone-cyanohydrin process which relies on acetone produced from petrochemically derived benzene and propylene. Styrene is produced through the highly energy intensive chemocatalytic dehydrogenation of ethylbenzene, which is also derived from petrochemical benzene. In order to improve the sustainability of this sector, biological routes of production are urgently needed. The production of both molecules is complicated by the toxicity of styrene and methacrylate esters such as butyl methacrylate (BMA) to the producing cells. In order to characterise the intrinsic suitability of Pseudomonas species as production hosts, the exogenous BMA tolerance of a number of Pseudomonas species including an environmental isolate was compared to E. coli. This established that Pseudomonas species (including P. putida) are innately highly BMA tolerant and capable of growing in a biphasic mixture of media and BMA at saturating BMA concentrations while E. coli is not. In order to identify the wild BMA tolerant Pseudomonas, a high quality, contiguous, closed genome was produced and characterised. Ultimately, P. putida was chosen for further characterisation. Through RNA-seq of BMA, ethylbenzene, and styrene exposed P. putida, we identified a suite of highly upregulated transcriptional regulators and RND efflux pumps. We subsequently generated deletion mutants in these systems and identified that deletion of a poorly characterised efflux pump (PP_1516/PP_1517) significantly reduced BMA tolerance and identified that deletion of the transcriptional repressor ttgR improved styrene tolerance due to the derepression of the ttgABC RND efflux pump. Through transposon sequencing, we screened the solvent tolerance of a library of P. putida transposon mutants, identifying 259 candidate genes for deletion/upregulation/retention for the development of a robust P. putida BMA production host.
- Advisor / supervisor
- Tucker, Nicholas
- Resource Type
- Note
- Previously held under moratorium from 16th June 2022 until 16th June 2025.
- DOI
Relations
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PDF of thesis T16645 | 2023-08-23 | Public | Download |