Thesis

The industrial microbiome as a resource for biotechnology : identification and isolation of butyl methacrylate tolerant pseudomonas species for enhanced sustainable bio-production of methacrylate esters

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17305
Person Identifier (Local)
  • 202068438
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • The increased demand for sustainable acrylic plastic has led to interest in developing a fermentation process to produce the monomers used to make this versatile compound. This would provide an alternative to the current finite and environmentally damaging petrochemical processes. Butyl Methacrylate (BMA) is an appealing precursor to these monomers. The current bottleneck for the fermentation of BMA is toxicity; its hydrophobic properties, which make it ideal for extraction from a bioreactor, are toxic to microorganisms interfering with cell membranes. Pseudomonas species, known for thriving in industrially contaminated sites, provide a potential solution to this bottleneck. By targeting industrial environments heavily contaminated with hydrophobic solvents, we isolated Pseudomonas species with tolerance to BMA. The most tolerant isolate, P. putida, held a plasmid, which significantly contributed to conferring tolerance to BMA. The primary tolerance mechanism was an RND(resistance-nodulation-division) eQlux pump, which when transferred to P. putida KT2440, significantly increased its tolerance to BMA. Features of this RND eQlux pump were used to identify other eQlux pumps which may also confer tolerance to hydrophobic compounds. To oQer additional tolerance and intracellular protection from BMA, we also used unsupervised machine learning to sort compounds produced by biosynthetic gene clusters (BGC) and BMA. The natural compound FR900098 an antimalarial candidate with a similar electrostatic profile to BMA was identified, and the corresponding resistance gene frbF associated with the BGC was cloned into a high-copy plasmid with a constitutive promotor. When transformed into P. putida KT2440, this resulted in a significant increase in BMA tolerance. In summary, the newly isolated P. putida strain (RCS09A) may be advantaged as an BMA production strain, due to its intrinsic tolerance to this hydrophobic molecule and a candidate for engineering for production. Alternatively, the identified RND eQlux pump and the resistance protein may be used to ‘upgrade’ the tolerance of other potential BMA production strains. Improved BMA tolerance could lead to increased product titres and lower the costs of the sustainable fermentation process.
Advisor / supervisor
  • Tucker, Nick
  • Cartman, Stephen
  • Tedman, Grace
  • Pritchard, Leighton
Resource Type
DOI
Date Created
  • 2024

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