METABOLOMICS: A Tool to Assess the Physiological Response of Pseudomonas Strains to Environmental Changes

Research output: Book/ReportPh.D. thesis – Annual report year: 2018Research

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Qualitative and quantitative analysis of intracellular metabolites is a valuable approach for characterizing and understanding the biochemical processes in cellular systems. Their composition and level represent the molecular phenotype of an organism in response to genetic or environmental conditions. However, analysis and quantification of intracellular metabolites is a challenging task due to their high turnover rates and chemical diversity. Thus, absolute quantification of intracellular metabolite levels requires well-validated sampling techniques that instantaneously stop the metabolic activity of the cell to avoid change in concentration of those metabolites. Furthermore, the analytical tools applied should be capable to cope with the large number of metabolites to be analyzed and the complex matrix
in the samples. The general aim of this thesis was to perform detailed physiological and omics-level characterization of Pseudomonas taiwanensis VLB120 and mutant strains constructed for biofuel production. In order to achieve the goal of the study, the first step was the development of a metabolomics work flow which was mainly focused on sample preparation as it is one of the fundamental and important steps in such studies. The developed technique was then applied to quantify the key intracellular metabolites of P. taiwanensis VLB120 in order to understand how genetic manipulations and environmental changes,influence the level of intracellular metabolites in Pseudomonas. In Paper I, new quenching/extraction techniques (cold ethanol, boiling ethanol, cold methanol: acetonitrile: water mix and hot water) were introduced that combines both quenching and extraction procedures in one single step. Their efficiency was evaluated based on energy charge (EC) ratio, recovery of the metabolites and experimental reproducibility. Based on those evaluation criteria, a fast filtration system followed by boiling ethanol quenching/extraction proved to work well for P. taiwanensis VLB120 metabolome analysis. Applying this technique, more than 100 intracellular metabolites were quantified for P. taiwanensis VLB120. These metabolites are not representative of the entire metabolome of P. taiwanensis VLB120, however, they have an essential role in central metabolism. The main chemical classification of these metabolites includes sugars phosphates, amino acids, organic acids, redox cofactors, nucleosides/bases and nucleotides.
In Paper II, the developed metabolomics tools were applied to evaluate the metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate derived inhibitors. P. taiwanensis responded to these inhibitors in various ways including detoxification, efflux, repair and tolerance to protect themselves against harsh environmental conditions. These lead the strain to go through metabolic rearrangement to generate more cellular energetics (e.g. adenosine triphosphate, ATP) and redox carrier (e.g. nicotinamide adenine dinucleotide phosphate-oxidase, NADPH) in an effort to mitigate the stress imposed by inhibitors. The data presented in this thesis show that the method developed during this PhD study was successfully applied for multi-targeted analysis of different classes of intracellular metabolites. The best-suited method was further applied to understand and confer tolerance to stress through genetic engineering. The work presented in this PhD thesis has shown to be a valuable addition to the “omics” tools used to reveal key information regarding metabolism and regulation in biological systems.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherNovo Nordisk Foundation Center for Biosustainability
Number of pages153
Publication statusPublished - 2017

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