Discovery and characterization of new Ene-reductases

Seven new putative ene-reductases (ERs or OYEs) have been identified and selected using bioinformatics tools from different organisms: Galdieria sulphuraria (GsOYE), Chroococcidiopsis thermalis (CtOYE), Chloroflexus aggregans (CaOYE), Botryotinia fuckeliana (BfOYE1 and BfOYE4) and Aspergillus niger (AnOYE2 and AnOYE8). Based on most updated literature, both the photosynthetic organisms (Galdieria, Chroococcidiopsis and Chloroflexus) and the fungi (Botryotinia and Aspergillus) result very interesting sources for ERs, that have not been exploited until now. The cloning and expression strategy used was the same for all the seven putative sequences. After a first trial of expression in E. coli BL21(DE3 good over-expression and solubility were obtained for GsOYE, CtOYE, CaOYE and BfOYE1 while for the other three proteins further optimization was necessary. To overcome the low solubility of AnOYE2 and AnOYE8 and the low production of BfOYE4 many strategies have been performed such as lowering the temperature of expression and using chaperons, but also trying other expression hosts (i.e. P. pastoris). A biocatalytic characterization was carried out for all proteins. Once verified their activity as ene-reductases in vitro, a steady-state study was performed in order to obtain the kinetics parameters. For two enzymes, GsOYE and CtOYE, bioconversions were set-up at the Biocatalysis laboratories of Graz University headed by Prof. Kurt Faber, a main expert of biocatalysis and also of ene-reductases. Finally, a biochemical characterization was carried on in order to determine the thermal stability, pH tolerance and also the three-dimensional structure of the newly discovered enzymes. Moreover, their application for the hydride-independent isomerization of non-activated C=C-bonds and subsequent reduction is also discussed and the efforts in elucidating this new reactivity are shown in detail. The work presented in this thesis lead to the discovery of new ERs enlarging the possibility to find out novel and promising biocatalysts for C=C-bond bioreduction but also for other unexpected biocatalytic reactivities.