title: Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli.
abstract: Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Howard
authors.forename: Sabine
authors.initials: S
authors.lastname: Middelhaufe
authors.forename: Karen
authors.initials: K
authors.lastname: Moore
authors.forename: Christoph
authors.initials: C
authors.lastname: Edner
authors.forename: Dagmara M
authors.initials: DM
authors.lastname: Kolak
authors.forename: George N
authors.initials: GN
authors.lastname: Taylor
authors.forename: David A
authors.initials: DA
authors.lastname: Parker
authors.forename: Rob
authors.initials: R
authors.lastname: Lee
authors.forename: Nicholas
authors.initials: N
authors.lastname: Smirnoff
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Aves
authors.forename: John
authors.initials: J
authors.lastname: Love
mesh_terms: D000473:Alkanes / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology; D056804:Biofuels; D001709:Biotechnology / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D027424:Cinnamomum / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000737:chemistry*; D005818:Genetic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D046937:Nostoc / Q000201:enzymology; D020637:Photorhabdus / Q000201:enzymology; D058615:Synthetic Biology / Q000379:methods
chemical_list: D000473:Alkanes; D056804:Biofuels; D005230:Fatty Acids, Nonesterified; D002244:Carbon
keywords: branched fatty acid biosynthesis; lux genes; metabolic engineering; synthetic biology