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Microbial production of short-chain alkanes

Abstract

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 β-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-ACPs2, 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 biosynthesis3. A modified thioesterase4 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 (adhEmut)6.

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Figure 1: Metabolic engineering of E. coli for the production of short-chain alkanes.
Figure 2: Effects of three types of acyl-ACP thioesterases and ‘TesA(L109P) on free fatty acid production.
Figure 3: GC–MS profile of fermentation products.

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Acknowledgements

We would like to thank Y. H. Lee for her assistance in cloning work and S. J. Choi for performing the fermentation experiments for checking reproducibility. This work was supported by the Advanced Biomass Research and Development Center of Korea (ABC-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF). Systems metabolic engineering work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) by MSIP through NRF.

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S.Y.L. conceived and supervised the project. Y.J.C. performed all experiments and analysed the data. Y.J.C. and S.Y.L. wrote the manuscript together. Both authors approved the final manuscript.

Corresponding author

Correspondence to Sang Yup Lee.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1-11, Supplementary Tables 1-4 and additional references. This file was replaced on 7 August 2014 to correct the primer sequence for the amplification of the ACR gene in Supplementary Table 4. (PDF 2190 kb)

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Choi, Y., Lee, S. Microbial production of short-chain alkanes. Nature 502, 571–574 (2013). https://doi.org/10.1038/nature12536

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