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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Han, L., Lobo, S. & Reynolds, K. A. Characterization of β-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis. J. Bacteriol. 180, 4481–4486 (1998)
Heath, R. J. & Rock, C. O. Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli. J. Biol. Chem. 271, 1833–1836 (1996)
Nunn, W. D., Giffin, K., Clark, D. & Cronan, J. E., Jr Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 154, 554–560 (1983)
Lo, Y. C., Lin, S. C., Shaw, J. F. & Liaw, Y. C. Substrate specificities of Escherichia coli thioesterase I/protease I/lysophospholipase L1 are governed by its switch loop movement. Biochemistry 44, 1971–1979 (2005)
Zhang, F., Carothers, J. M. & Keasling, J. D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature Biotechnol. 30, 354–359 (2012)
Holland-Staley, C. A., Lee, K., Clark, D. P. & Cunningham, P. R. Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid. J. Bacteriol. 182, 6049–6054 (2000)
Peralta-Yahya, P. P., Zhang, F., del Cardayre, S. B. & Keasling, J. D. Microbial engineering for the production of advanced biofuels. Nature 488, 320–328 (2012)
Lennen, R. M., Braden, D. J., West, R. A., Dumesic, J. A. & Pfleger, B. F. A process for microbial hydrocarbon synthesis: overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol. Bioeng. 106, 193–202 (2010)
Schirmer, A., Rude, M. A., Li, X., Popova, E. & del Cardayre, S. B. Microbial biosynthesis of alkanes. Science 329, 559–562 (2010)
Harger, M. et al. Expanding the product profile of a microbial alkane biosynthetic pathway. ACS Synthet. Biol. 2, 59–62 (2013)
Howard, T. P. et al. Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc. Natl. Acad. Sci. USA 110, 7636–7641 (2013)
Altin, O. & Eser, S. Carbon deposit formation from thermal stressing of petroleum fuels. Am. Chem. Soc. Div. Fuel Chem. 49, 764–766 (2004)
Atsumi, S., Hanai, T. & Liao, J. C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008)
Choi, Y. J., Park, J. H., Kim, T. Y. & Lee, S. Y. Metabolic engineering of Escherichia coli for the production of 1-propanol. Metab. Eng. 14, 477–486 (2012)
Gary, J. H. & Handwerk, G. E. Petroleum Refining: Technology and Economics 4th edn (Marcel Dekker, 2001)
Naggert, J. et al. Cloning, sequencing, and characterization of Escherichia coli thioesterase II. J. Biol. Chem. 266, 11044–11050 (1991)
Steen, E. J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010)
Pollard, M. R., Anderson, L., Fan, C., Hawkins, D. J. & Davies, H. M. A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica. Arch. Biochem. Biophys. 284, 306–312 (1991)
Jing, F et al. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochem. 12, 44 (2011)
Zheng, Y et al. Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase. Biotechnol. Biofuels 5, 76 (2012)
Torella, J. P Tailored fatty acid synthesis via dynamic control of fatty acid elongation. Proc. Natl Acad. Sci. USA 110, 11290–11295 (2013)
Tsay, J. T., Oh, W., Larson, T. J., Jackowski, S. & Rock, C. O. Isolation and characterization of the β-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12. J. Biol. Chem. 267, 6807–6814 (1992)
Na, D. et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnol. 31, 170–174 (2013)
Zhang, H., Wang, P. & Qi, Q. Molecular effect of FadD on the regulation and metabolism of fatty acid in Escherichia coli. FEMS Microbiol. Lett. 259, 249–253 (2006)
Aarts, M. G., Keijzer, C. J., Stiekema, W. J. & Pereira, A. Molecular characterization of the CER1 gene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell 7, 2115–2127 (1995)
McNevin, J. P., Woodward, W., Hannoufa, A., Feldmann, K. A. & Lemieux, B. Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana. Genome 36, 610–618 (1993)
Reiser, S. & Somerville, C. Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase. J. Bacteriol. 179, 2969–2975 (1997)
Bernard, A. et al. Reconstitution of plant alkane biosynhtesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell 24, 3106–3118 (2012)
Bourdenx, B Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stress. Plant Physiol. 156, 29–45 (2011)
Sambrook, J. R. D. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001)
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)
Yuan, L. Z., Rouviere, P. E., Larossa, R. A. & Suh, W. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab. Eng. 8, 79–90 (2006)
Palmeros, B. et al. A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene 247, 255–264 (2000)
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.
Author information
Authors and Affiliations
Contributions
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
Ethics declarations
Competing interests
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)
Rights and permissions
About this article
Cite this article
Choi, Y., Lee, S. Microbial production of short-chain alkanes. Nature 502, 571–574 (2013). https://doi.org/10.1038/nature12536
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature12536
This article is cited by
-
State-of-art engineering approaches for ameliorated production of microbial lipid
Systems Microbiology and Biomanufacturing (2024)
-
Advances in the optimization of central carbon metabolism in metabolic engineering
Microbial Cell Factories (2023)
-
Genomic insights into cryptic cycles of microbial hydrocarbon production and degradation in contiguous freshwater and marine microbiomes
Microbiome (2023)
-
Biosynthesis pathways of expanding carbon chains for producing advanced biofuels
Biotechnology for Biofuels and Bioproducts (2023)
-
Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression
Applied Microbiology and Biotechnology (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.