O'Sullivan, Lisa; (2000) Pathway engineering of the toluene degradation pathway of Pseudomonas putida mt-2. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text U643596.pdf Download (21MB) | Preview

Abstract

The TOL meta-cleavage pathway of Pseudomonas putida mt-2 has previously been mathematically modelled by Regan et al (1994), in order to simulate flux through the pathway. Cells carrying the entire meta-cleavage pathway encoded on plasmid pQR150, and a truncated version of the pathway encoded on plasmid pQR226, were studied. The predicted flux-controlling step, the xylXYZ encoded toluate dioxygenase was overexpressed with either the entire or truncated pathway, in E. coli JM107 and growth and enzyme activities investigated. The amplification of toluate dioxygenase lead to higher enzyme activities, specifically of the key pathway enzyme, catechol 2,3-dioxygenase, suggesting an increase of flux through the pathway. Overexpression of the xylZ encoded reductase subunit of toluate dioxygenase suggested the need for xylZ transcription on the same plasmid as xylXY for the enzyme to play its proposed role in catechol 2,3-dioxygenase reactivation. An investigation into the effect of benzoate concentration on cell growth and TOL pathway metabolism was carried out. Cells expressing amplified toluate dioxygenase had an increased tolerance and benzoate metabolism. All strains carrying the TOL pathway increased intermediate production and enzyme activities with increasing benzoate concentration, despite a reduction in growth. This finding is in agreement with Regan's model (1991). However, there were threshold values for each strain over which pathway metabolism shut down. The effect of pH on benzoate toxicity and pathway flux was also studied. Overall, cells grown in alkali media did not appear to have increased benzoate tolerance. The amplified toluate dioxygenase strain appeared to have an initial increase in flux at pH 6.0 and it was suggested that this was due to a greater influx of benzoate into the cell. Cell morphology was compared between growth in complex and minimal media because of poor growth and pathway flux for all strains in minimal media. E. coli JM107 became filamentous in minimal media. In order to broaden the substrate range of the meta-cleavage pathway and to produce novel muconolactones, a hybrid pathway was formed. CatA from the β-ketoadipate pathway of Acinetobacter calcoaceticus was amplified and cloned into pQR227 (xylXYZLT) to form pQR234. The reasons for poor expression of catA in this construct are discussed.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item