Aliphatic hydrocarbons are excellent biofuel targets, as they are already predomi­nant components of petroleum-based gasoline and diesel fuels and would be com­patible with existing engines and fuel distribution systems. Recently, genes for alkene biosynthesis (oleABCD) have been identified in multiple bacteria and the functions of these genes elucidated in in vivo and in vitro studies (e. g., [1]).

The OleABCD proteins catalyze the condensation of fatty acids to long-chain alkenes and this production is enhanced in E. coli by the overproduction of fatty acids. To produce long-chain alkenes in R. eutropha, fatty acid overproduction will be engineered into R. eutropha using techniques that have been used successfully in other proteobacteria [1, 21].

2.2.1 Farnesene

The isoprenoid pathway represents an important source of advanced biofuel precur­sors such as farnesene. Chemical hydrogenation of farnesene produces farnesane, which can serve as a diesel fuel. All terpenoids originate from the same universal precursors (isopentenyl pyrophosphate [IPP] and its isomer dimethylallyl pyrophos­phate [DMAPP]), which can be generated through two known biosynthetic path — ways—the mevalonate-dependent (MEV) isoprenoid pathway mostly found in eukaryotes and the deoxyxylulose 5-phosphate (DXP) pathway found in most prokaryotes. The R. eutropha genome encodes the DXP pathway, which generates the precursor molecules IPP and DMAPP demonstrated to be essential in prokary­otes for the prenylation of tRNAs and the synthesis of farnesyl pyrophosphate (FPP), which is used for quinone and cell wall biosynthesis [15]. While farnesene may be produced in R. eutropha through the manipulation of its native DXP path­way, the tight regulation of essential metabolites produced through this route may pose a significant challenge in achieving reasonable titers. For optimal production of isoprenoid-based fuel molecules, we therefore propose the incorporation of the MEV pathway, which we expect to be unregulated by this host. We will synthesize and express genes originating from distinct prokaryotic and eukaryotic sources to enable production of a-farnesene in the R. eutropha strains.