Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. 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)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 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)

    Article  CAS  Google Scholar 

  3. 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)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 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)

    Article  CAS  Google Scholar 

  5. 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)

    Article  CAS  Google Scholar 

  6. 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)

    Article  CAS  Google Scholar 

  7. 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)

    Article  ADS  CAS  Google Scholar 

  8. 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)

    Article  CAS  Google Scholar 

  9. Schirmer, A., Rude, M. A., Li, X., Popova, E. & del Cardayre, S. B. Microbial biosynthesis of alkanes. Science 329, 559–562 (2010)

    Article  ADS  CAS  Google Scholar 

  10. Harger, M. et al. Expanding the product profile of a microbial alkane biosynthetic pathway. ACS Synthet. Biol. 2, 59–62 (2013)

    Article  CAS  Google Scholar 

  11. 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)

    Article  ADS  CAS  Google Scholar 

  12. Altin, O. & Eser, S. Carbon deposit formation from thermal stressing of petroleum fuels. Am. Chem. Soc. Div. Fuel Chem. 49, 764–766 (2004)

    CAS  Google Scholar 

  13. Atsumi, S., Hanai, T. & Liao, J. C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008)

    Article  ADS  CAS  Google Scholar 

  14. 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)

    Article  Google Scholar 

  15. Gary, J. H. & Handwerk, G. E. Petroleum Refining: Technology and Economics 4th edn (Marcel Dekker, 2001)

    Book  Google Scholar 

  16. Naggert, J. et al. Cloning, sequencing, and characterization of Escherichia coli thioesterase II. J. Biol. Chem. 266, 11044–11050 (1991)

    CAS  PubMed  Google Scholar 

  17. Steen, E. J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010)

    Article  ADS  CAS  Google Scholar 

  18. 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)

    Article  CAS  Google Scholar 

  19. 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)

    Article  Google Scholar 

  20. 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)

    Article  Google Scholar 

  21. Torella, J. P Tailored fatty acid synthesis via dynamic control of fatty acid elongation. Proc. Natl Acad. Sci. USA 110, 11290–11295 (2013)

    Article  ADS  Google Scholar 

  22. 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)

    CAS  PubMed  Google Scholar 

  23. Na, D. et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnol. 31, 170–174 (2013)

    Article  CAS  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. 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)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 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)

    Article  CAS  Google Scholar 

  27. 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)

    Article  CAS  Google Scholar 

  28. 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)

    Article  CAS  Google Scholar 

  29. 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)

    Article  Google Scholar 

  30. Sambrook, J. R. D. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001)

    Google Scholar 

  31. 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)

    Article  ADS  CAS  Google Scholar 

  32. 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)

    Article  CAS  Google Scholar 

  33. 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)

    Article  CAS  Google Scholar 

Download references

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

Authors

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

Correspondence to Sang Yup Lee.

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)

PowerPoint slides

Rights and permissions

Reprints 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12536

This article is cited by

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.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research