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Univ. of Exeter team engineers unique biological pathway for the production of diesel range hydrocarbons by E. coli

Howrd
Hydrocarbons produced by cells expressing the synthetic alkane pathway (CEDDEC) or the cyanobacterial alkane pathway (AR and AD from N. punctiforme) without modifications to the fatty acid pool. n = 6 biological replicates; error bars represent SE of mean. Howard et al. Click to enlarge.

A team from the University of Exeter (UK), with support from Shell Technology Centre Thorton, has modified strains of E. coli bacteria to produce “petroleum-replica” hydrocarbons in the diesel range. While the technology still faces many significant commercialization challenges, the resulting drop-in fuel is almost identical to conventional diesel fuel and so does not need to be blended with petroleum products as is often required by biodiesels derived from plant oils.

In an open access paper on their work published in the Proceedings of the National Academies of Science, the researchers note that their work—rather than reconstituting existing metabolic routes to alkane production found in nature—demonstrated the ability to design and to implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.

Biogenic alkanes are found throughout nature, but not in the required form for direct replacement fuels; new pathways for the biological production of different alkanes must therefore be developed. The only genetically characterized alkane biosynthesis pathways are the production of very long chain alkanes (>C28) by Arabidopsis thaliana and the production of pentadecane (C15:0) and heptadecane (C17:0) by cyanobacteria. The cyanobacterial alkane pathway has also been biochemically characterized: fatty acyl-acyl carrier protein (ACP) is reduced to fatty aldehyde (Cn) by acyl-ACP reductase (AR), which is then decarbonylated to an alkane (Cn-1) by fatty aldehyde decarbonylase (AD), primarily producing heptadecene (C17:1) in Escherichia coli and releasing formate. To advance biologically synthesized alkanes further into the fuel market, they must meet the technical requirements demanded of retail fuels, not the requirements of the source organism.

The objective of the present study was to address the problem of direct fossil fuel replacements through the de novo design and assembly of synthetic metabolic pathways, with the aim of producing linear and branched-chain alkanes and alkenes of variable, but specified, Cn. To achieve this aim, we engineered E. coli cells to use free FAs [fatty acids] directly, rather than the FA thioesters used by the native cyanobacterial pathway. Exploiting free FAs as entry substrates for alkane synthesis meant that predictable alterations to the hydrocarbon output of the cells was possible by modifications to the free FA pool, either by additional pathway engineering or by exogenous supplementation of the media. Using this strategy, E. coli expressing different molecular modules produced linear tridecane (C13:0), tridecene (C13:1), pentadecane, pentadecene (C15:1), hexadecane (C16:0), hexadecene (C16:1), heptadecane, heptadecene, and branched tridecane and pentadecane, thereby replicating the chemical and structural requirements of retail diesel hydrocarbons commonly used in temperate climates. The results thus provide proof of principle for the metabolic production of industrially relevant direct petroleum-replica hydrocarbons.

—Howard et al.

They targeted conversion of free FAs, rather than fatty acyl-ACP, to alkanes for three main reasons: first, exploiting the FA pool would provide a mechanism to direct alkane biosynthesis to different Cn alkanes; second, FAs are more abundant in cells than fatty acyl-ACP; and third, the FA pool can be substantially increased through genetic manipulation.

One of the most important characteristics of the genetic modules described here is that there is no “blend wall” for the biologically generated fossil-fuel replicas synthesized. The identification of limitations in conversion of aldehydes to alkanes and of an alternative metabolic sink provides a focus for future engineering strategies: these may involve removing competing reactions, generating an intracellular environment in which the decarbonylase can more efficiently use the fatty aldehydes generated by the first step of the pathway or improving the efficiency of the decarbonylase itself.

The results presented in this article, although at a very early stage in product development, contribute toward the goals of advanced biofuels by providing metabolic pathways for the production of industrially relevant, petroleum-replica fuel molecules.

—Howard et al.

This work was supported by a grant from Shell Research Ltd and a Biotechnology and Biological Sciences Research Council (BBSRC) Industry Interchange Partnership Grant.

Resources

  • Thomas P. Howard, Sabine Middelhaufe, Karen Moore, Christoph Edner, Dagmara M. Kolak, George N. Taylor, David A. Parker, Rob Lee, Nicholas Smirnoff, Stephen J. Aves, and John Love (2013) Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. PNAS 2013 doi: 10.1073/pnas.1215966110

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