Metabolic Engineering of S. Cerevisiae for the Production of Propane

The goal of this experiment was to produce propane from Saccharomyces cerevisiae through a novel biosynthetic genetic pathway using genes KivD and ADO from Lactococcus lactis and Prochlorococcus marinus respectively. These genes branch off of S. cerevisiae’s metabolism from ketoisovalerate in the valine pathway. With increasing fuel prices worldwide and the possibility of a future energy crisis, there has been a strong shift towards developing renewable fuel systems from microorganisms. Propane, in particular, is an advantageous substance due to its diverse uses in motor vehicles, furnace heaters, and cooking fuels. Introduction of propane synthesis into S. cerevisiae helps these efforts by developing a high-producing fuel strain that can easily be grown to industrial sizes. A metabolic model of this organism was examined to determine any gene knockouts that would direct more of S. cerevisiae’s metabolism towards the production of propane. Further, production of propane in S. cerevisiae was compared for strains with and without the additional insertion of various valine pathway genes isolated from Penicillium chrysogenum. The high producing nature of the P. chrysogenum valine pathway was exploited by the introduction of eighteen different gene sequence iterations of the pathway into Saccharomyces cerevisiae cells. The pathway of P. chrysogenum was suggested to be high producing as L-valine is a key precursor to penicillin, an evolutionarily significant molecule to this organism. Though P. chyrsogenum is predicted to have a higher production of propane, it is much more difficult to grow at industrially relevant levels because it is multi-cellular and aggregates in media. S. cerevisiae was selected as the cellular host because it has a well characterized genome and metabolism, and it is much more easily grown to industrial standards. Yeast assembly methods were used to introduce the different iterations of the P. chrysogenum valine pathway genes into S. cerevisiae each including a Nourseothricin selection gene as well. This included a high and low producing strain for pathways using one of three genes involved in the expression of AHAS and ilvD proteins (AHAS I, AHAS II, AHAS III; ilvD I, ilvD II, ilvD III). Additional analyses were performed to determine which of these genes was responsible for regulation of each enzyme’s activity. Propane production of individual strains was quantified and compared to determine the efficiency of each strain. Once the highest producing strain has been selected, metabolic engineering techniques, such as increasing strain tolerance through serial subculturing and enhancing culture conditions, will be performed to optimize efficiency in this pathway while it is grown up to industrially relevant levels. Optimization of propane production in S. cerevisiae for use in industrial processes is an advantageous way to obtain valuable biofuels as the world faces an energy crisis.