Pseudomonas aeruginosa synthesises heterogeneous mixtures of mono and di-rhamnolipid (RL) biosurfactants. The complex genetic and metabolic regulation surrounding RL biosynthesis is one of the major hurdles for the large scale production of these metabolites. To date there is still a lack of quantitative understanding of the interaction between the metabolic and regulatory networks governing RL biosynthesis in P. aeruginosa. The initial part of the thesis details the first systematic validation of reference gene stability under nutrient limiting conditions for the development of a reliable and accurate RT-qPCR method specific for RL biosynthetic gene expression. A specific UPLC-MSMS method was simultaneously developed and validated for the quantitative determination of RL yield and congener distribution in P. aerllginosa. This is the first report of individual RL quantification using UPLC-MSMS. The second part of the thesis investigated the effect of hydrophilic and hydrophobic carbon substrates on RL production. Consistent with such a high level of regulatory complexity for P. aerllginosa, transcriptional analysis of the RL biosynthetic genes revealed a highly conserved gene expression profile. RL biosynthetic genes rhlAB (responsible for mono-rhamnolipid synthesis) were not induced until early stationary phase when growth had significantly slowed down. Expression of rhlC (responsible for di-rhamnolipid synthesis) was induced during later-stationary phase after maximum up-regulation of rhlAB expression. Choice of carbon substrate for RL production significantly influenced the expression level and timing of RL biosynthetic genes, with hydrophobic substrates inducing the highest overall up-regulation of RL genes. UPLC-MSMS revealed a conserved RL congener distribution , mono-RLs were more abundant during early-stationary phase followed by a predominance of di-rhamnolipids accumulating during mid to late stationary phases. RL composition was regulated by the selective incorporation of CIO ~-hydroxy fatty acids into RLs with Rha-Rha-C1o-C IO and RhaC10- C IO the most abundant congeners produced irrespective of carbon substrate metabolised. UPLC-MSMS also showed that inactivation of rhlC led to the production of mono-RL only congeners in yields and composition comparable to wild-type P. aeruginosa. The data suggests that expression of rhlA B is not only cell density regulated but also stringently regulated by nutritional cues (i.e. carbon source and macronutrient limitation). Thus de novo RL biosynthesis is prudently regulated at the transcriptional level, the regulation of rhlAB is the rate limiting step which dictates the extracellular RL profile. From a biotechnological point of view, this prudent metabolic regulation may explain why RLs are synthesised in such relative low yields and is perhaps a mechanism by which Pseudomonas aerllginosa has evolved to adapt to nutrient limiting environments. This thesis presents a systems metabolic engineering approach for quantitatively assessing RL production under nutrient limiting conditions
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:650312 |
| Date | January 2014 |
| Creators | Rudden, Michelle |
| Publisher | Ulster University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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