Microbial production of short-chain alkanes

Increasing concerns about limited fossil fuels and global environmental problems have focused attention on the need to develop sustainable biofuels from renewable resources. Although microbial production of diesel has been reported, production of another much in demand transport fuel, petrol (gasoline), has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway. First, the beta-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo. To increase the formation of short-chain fatty acids suitable for subsequent conversion to SCAs in vivo, the activity of 3-oxoacyl-ACP synthase (FabH)(1), which is inhibited by unsaturated fatty acyl-ACPs(2), was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene; deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for unsaturated fatty acids biosynthesis(3). A modified thioesterase(4) was used to convert short-chain fatty acyl-ACPs to the corresponding FFAs, which were then converted to SCAs by the sequential reactions of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l(-1) of SCAs consisting of nonane (327.8 mg l(-1)), dodecane (136.5 mg l(-1)), tridecane (64.8 mg l(-1)), 2-methyl-dodecane (42.8 mg l(-1)) and tetradecane (8.9 mg l(-1)), together with small amounts of other hydrocarbons. Furthermore, this platform strain could produce short-chain FFAs using a fadD-deleted strain, and short-chain fatty esters by introducing the Acinetobacter sp. ADP1 wax ester synthase (atfA)(5) and the E. coli mutant alcohol dehydrogenase (adhE(mut))(6).