Letter

Microbial production of fatty-acid-derived fuels and chemicals from plant biomass

Received:
Accepted:
Published online:

Abstract

Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals1. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective ‘consolidated bioprocesses’2. 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.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    et al. Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechnol. 26, 375–381 (2008)

  2. 2.

    , , & Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, 577–583 (2005)

  3. 3.

    , , , & Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc. Natl Acad. Sci. USA 103, 11206–11210 (2006)

  4. 4.

    & New microbial fuels: a biotech perspective. Curr. Opin. Microbiol. 12, 274–281 (2009)

  5. 5.

    , , & Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol. Rev. 57, 522–542 (1993)

  6. 6.

    & Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action. J. Bacteriol. 176, 2814–2821 (1994)

  7. 7.

    & Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J. Biol. Chem. 270, 4216–4219 (1995)

  8. 8.

    , & Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab. Eng. 10, 333–339 (2008)

  9. 9.

    , , & Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana. Plant J. 9, 167–172 (1996)

  10. 10.

    , & Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008)

  11. 11.

    et al. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb. Cell Fact. 7, 36 (2008)

  12. 12.

    Oil. Market Report, International Energy Agency〉 (2008 01 16)

  13. 13.

    , & Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152, 2529–2536 (2006)

  14. 14.

    & The changing world of oleochemicals. Palm Oil Developments 44, 15–28 (2006)

  15. 15.

    & Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions. J. Biol. Chem. 279, 37789–37797 (2004)

  16. 16.

    , , , & Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed. Plant Physiol. 122, 635–644 (2000)

  17. 17.

    & 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)

  18. 18.

    “Designer” biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22, 1358–1364 (2008)

  19. 19.

    , , , & Genetic engineering of ethanol production in Escherichia coli. Appl. Environ. Microbiol. 53, 2420–2425 (1987)

  20. 20.

    , , , & Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes. Microbiology 150, 2257–2266 (2004)

  21. 21.

    & The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region. J. Bacteriol. 172, 2408–2412 (1990)

  22. 22.

    , , & Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnol. Bioeng. 101, 587–601 (2008)

  23. 23.

    et al. High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One 4, e4489 (2009)

  24. 24.

    & One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

  25. 25.

    , & Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J. Bacteriol. 179, 6228–6237 (1997)

  26. 26.

    & DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res. 30, e43 (2002)

  27. 27.

    & Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 4, 251–256 (2007)

  28. 28.

    , & Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)

  29. 29.

    , , & Gas–liquid chromatographic method for analysing complex mixtures of fatty acids including conjugated linoleic acids (cis9trans11 and trans10cis12 isomers) and long-chain (n-3 or n-6) polyunsaturated fatty acids: application to the intramuscular fat of beef meat. J. Chromatogr. A 1110, 133–139 (2006)

Download references

Acknowledgements

E.J.S. was supported by the Tien Scholar Environmental Fellowship and the Synthetic Biology Engineering Research Center (SynBERC). Y.K. and G.B. were supported by a grant from LS9, Inc. (South San Francisco, California) through the University of California Discovery Grant program. This research was performed at the Joint BioEnergy Institute. We thank M. Rude with help on the manuscript and J. Cronan and the LS9 Scientific Advisory Board for technical insight and discussion.

Author Contributions E.J.S., Y.K., G.B., Z.H., A.S., A.M., S.B.d.C. and J.D.K. conceived of the experiments. E.J.S. and Y.K. constructed the strains and metabolic pathways for fatty-acid-derived products and performed the production experiments. LS9 engineered and evaluated FAEE and fatty alcohol producing strains for thioesterase evaluations. G.B. conceived, constructed and performed the xylan-metabolizing pathway growth experiments. E.J.S. and Y.K. constructed the xylan-metabolizing, fatty acid production strain and performed the production experiments. E.J.S., Y.K., A.S., S.B.d.C. and J.D.K. drafted the manuscript. All authors approved the final manuscript.

Author information

Author notes

    • Eric J. Steen
    •  & Yisheng Kang

    These authors contributed equally to this work.

Affiliations

  1. Joint BioEnergy Institute,

    • Eric J. Steen
    • , Yisheng Kang
    • , Gregory Bokinsky
    •  & Jay D. Keasling
  2. Synthetic Biology Engineering Research Center, 5885 Hollis Avenue, Emeryville, California 94608, USA

    • Eric J. Steen
    •  & Jay D. Keasling
  3. Departmient of Bioengineering,

    • Eric J. Steen
    •  & Jay D. Keasling
  4. QB3 Institute,

    • Yisheng Kang
    • , Gregory Bokinsky
    •  & Jay D. Keasling
  5. Department of Chemical Engineering, University of California, Berkeley, California 94720, USA

    • Jay D. Keasling
  6. LS9, Inc., 100 Kimball Way, South San Francisco, California 94080, USA

    • Zhihao Hu
    • , Andreas Schirmer
    • , Amy McClure
    •  & Stephen B. del Cardayre
  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jay D. Keasling

Authors

  1. Search for Eric J. Steen in:

  2. Search for Yisheng Kang in:

  3. Search for Gregory Bokinsky in:

  4. Search for Zhihao Hu in:

  5. Search for Andreas Schirmer in:

  6. Search for Amy McClure in:

  7. Search for Stephen B. del Cardayre in:

  8. Search for Jay D. Keasling in:

Competing interests

J.D.K. has financial interests in Amyris and LS9, both of which are involved in producing advanced biofuels. Z.H., A.S., A.M. and S.B.d.C. have a financial interest in LS9.

Corresponding authors

Correspondence to Stephen B. del Cardayre or Jay D. Keasling.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Tables 1-2, Supplementary Figures 1-3 with Legends and Supplementary References.

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.