Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective 'consolidated bioprocesses'. Fatty acids are composed of long alkyl chains and represent nature's 'petroleum', being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals. A more scalable, controllable and economic route to this important class of chemicals would be through the microbial conversion of renewable feedstocks, such as biomass-derived carbohydrates. Here we demonstrate the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars. Furthermore, we show engineering of the biodiesel-producing cells to express hemicellulases, a step towards producing these compounds directly from hemicellulose, a major component of plant-derived biomass.

Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.

Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.

Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1-nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleT JE ), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleT JE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in ␣-and/or ␤-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

For millennia, selective breeding, on the basis of biparental mating, has led to the successful improvement of plants and animals to meet societal needs. At a molecular level, DNA shuffling mimics, yet accelerates, evolutionary processes, and allows the breeding and improvement of individual genes and subgenomic DNA fragments. We describe here whole-genome shuffling; a process that combines the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes normally associated with conventional breeding. We show that recursive genomic recombination within a population of bacteria can efficiently generate combinatorial libraries of new strains. When applied to a population of phenotypically selected bacteria, many of these new strains show marked improvements in the selected phenotype. We demonstrate the use of this approach through the rapid improvement of tylosin production from Streptomyces fradiae. This approach has the potential to facilitate cell and metabolic engineering and provide a non-recombinant alternative to the rapid production of improved organisms.