Sophorolipids have been known for over 40 years as biological surface active compounds or surfactants. They can be produced by a selected number of yeast strains starting from renewable resources or even waste streams, dispose low ecotoxicity and are biodegradable. These features make them particularly attractive in our current society with growing environmental awareness. Millions of tons of surfactants are yearly used in a wide range of sectors (cleaning, chemical, textile, food and paper industry, cosmetics, personal and health care, agriculture, etc.), but the large majority of them is produced by chemical means. Especially in washing applications they inevitably end up in the environment where they are not always that readily degraded.Sophorolipids synthesized by the yeast Candida bombicola are one of the most promising biosurfactants; they are synthesized by nonpathogenic yeasts, can be produced at high concentrations and find applications in various sectors due to either their emulsifying, antimicrobial or other beneficial properties.Sophorolipids are composed of the disaccharide sophorose to which a hydroxy fatty acid is linked. The carbon chain length of these hydroxy fatty acids is limited to 16 to 18 carbon atoms. However, to increase the hydrophilic-lipophilic balance and the foaming capacities of the molecules, it would be interesting to obtain sophorolipids with a shorter fatty acid tail of 8 to 14 carbon atoms. These can on one hand be obtained by using unconventional hydrophilic carbon sources such as hydroxylated substrates or substrates with internal ester bonds which are post-fermentative modified.On the other hand, medium-chain sophorolipids can be obtained via the genetic engineering of the producing yeast strain. Since no such research has been reported till now, a transformation and selection system for C. bombicola was developed. In order to achieve this, the URA3 gene was isolated from the totally unknown C. bombicola genome. Several other genes were also isolated, among them the GAPD gene who’s promoter sequence can be used for (heterologous) expression of genes in C. bombicola, and the MFE-2 gene. The corresponding enzyme of this latter gene takes part in the β-oxidation pathway and knocking out of this gene resulted in improved medium-chain sophorolipid production on unconventional substrates.Cytochrome P450 monooxygenases play a crucial role in sophorolipid synthesis: they control the chain length of the sophorolipid hydroxy fatty acid tail when using conventional substrates. Several of these genes were isolated from the C. bombicola genome and two of them were assigned to new families. It is expected that not all of them take part in sophorolipid synthesis, but one cytochrome P450 monooxygenase shows a strong induction upon sophorolipid synthesis; it can therefore be suggested that this enzyme contributes to the formation of sophorolipids.This PhD-thesis presents the exciting first steps in the genetic engineering and in-depth study of the sophorolipid synthesis pathway of C. bombicola. The development of molecular tools and the use of unconventional substrates made it possible to produce medium-chain sophorolipids and gave better insights in the involved biochemical pathways.Furthermore, the presented work opens up perspectives for future and long term research on sophorolipid synthesis and C. bombicola.
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