(560f) Development of Alkene Biofuels

Goh, E., Joint BioEnergy Institute (JBEI)

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 C27monoketone. 
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.