Photosynthetic production of raw materials for jet fuel using cyanobacteria : In vivo study of recombinant oleate hydratase for isobutene production

Abstract: Aviation accounts for a significant part of the world’s CO2 emission from the transport sector, but sustainable jet fuels are not as developed or as commonly used as renewable fuels for road vehicles. Biofuels produced through photosynthesis in cyanobacteria are of interest to help solve the pressing issue of CO2 emissions and increased global warming. Isobutene, a volatile gas converted from isobutanol, could be assembled into sustainable jet fuel in a photo-biochemical system utilizing cyanobacteria. In this study, I examined the possibility of using metabolic engineering of Escherichia coli and the cyanobacterial strain Synechocystis PCC 6803 for enzymatic conversion of isobutanol to isobutene using recombinantly expressed oleate hydratase (OhyA), originating from four different organism. Plasmids containing the kivd gene, coding for the enzyme α-ketoisovalerate decarboxylase (Kivd) which synthesise isobutanol, and the ohyA gene were constructed and inserted into E. coli and Synechocystis. The inserted OhyA variants were successfully expressed and showed isobutanol production in Synechocystis, confirming functionality of Kivd. From the experimental setup and OhyA variants used in this thesis, in vivo production of isobutene from isobutanol was not detectable in E. coli or Synechocystis. These results indicate OhyA inactivity for the conversion of isobutanol to isobutene in vivo using this particular experimental setup. For future in vitro studies, three OhyA variants were successfully purified by affinity chromatography. Two of these variants showed FAD binding. The findings of this study will contribute to ongoing research on characterization of OhyA and cyanobacterial conversion of CO2 to isobutene for jet fuel production.