Pseudomonas putida as a microbial cell factory

Vinoth Wigneswaran

    Research output: Book/ReportPh.D. thesisResearch

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    The extensive use of fossil fuels has a severe influence on the environment. In order to reduce the dependency on these limited resources and to protect the environment substantial effort is being made to implement renewable resources. One part of this transition is to develop methods for sustainable production of chemicals, which can be achieved by microbial cell factories. The work presented in this PhD thesis elucidates the application of Pseudomonas putida as a microbial cell factory for production of the biosurfactant rhamnolipid. The rhamnolipid production was achieved by heterologous expression of the rhlAB operon from Pseudomonas aeruginosa using a synthetic promoter library in P. putida. Since rhamnolipid production is associated with difficulties in conventional bioreactors we have used biofilm encased P. putida to circumvent these problems. We show that biofilm can be used as a production platform for continuous production of rhamnolipids. A method for quantitative and qualitative analysis of the produced rhamnolipids was developed based on ultra performance liquid chromatography combined with high resolution mass spectrometry. This enabled detection of low levels of rhamnolipids. The applicability of glycerol as a substrate was also investigated. Since glycerol is a poor substrate adaptive evolution was made in order to improve the capabilities of P. putida to proliferate on glycerol. The evolved lineages all had significantly increased growth rate, enhanced cell density and reduced lag phase. The genomic alterations were identified by genome sequencing and revealed parallel evolution. Glycerol was also shown to be able to support biofilm growth and as a result of this it can be used as an alternative substrate for producing biochemicals in conventional and biofilm reactors. The use of biofilm as a production platform and the usage of glycerol as a feedstock show the potential of using microbial cell factories in the transition toward sustainable production of chemicals. Particularly, the applicability of biofilm as a production platform can emerge as a promising alternative for producing toxic biochemicals and for producing biochemicals which are difficult to cope in conventional bioreactors.
    Original languageEnglish
    PublisherDepartment of Systems Biology, Technical University of Denmark
    Number of pages122
    Publication statusPublished - 2016

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