Development of Alkene Biofuels

Aliphatic hydrocarbons are appealing targets for advanced cellulosic biofuels, as they are predominant components of petroleum-based gasoline and diesel fuels and thus would be compatible with existing engines and fuel distribution systems. Long-chain alkenes are useful as feedstocks that can be cracked to form diesel- or gasoline-range hydrocarbons.  We have studied alkene biosynthesis in the bacterium Micrococcus luteus, a close relative of which was reported in the 1960s to biosynthesize iso- and anteiso-branched, long-chain alkenes.  The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies.  In fact, until very recently, there were no known sequences for prokaryotic enzymes catalyzing the synthesis of non-isoprenoid aliphatic hydrocarbons (> C₂).  We have shown that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds).  Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C₂₇ monoketone.  Gas chromatography-time of flight (GC-TOF) mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis).  Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites.  We have proposed a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA.  Such activity is consistent with our data and with the homology of Mlut_13230 (OleA) to FabH, which catalyzes decarboxylative Claisen condensation during fatty acid biosynthesis.  We will discuss how understanding of the alkene biosynthetic pathway can lead to enhanced production.