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

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.

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Figure 1: Engineered pathways for production of fatty acid-derived molecules from hemicelluloses or glucose and depiction of the synthetic operons used in this study.
Figure 2: Total free fatty acid production and respective theoretical yield by engineered E. coli strains.
Figure 3: Engineered production of FAEEs and fatty alcohols with controlled chain length.
Figure 4: Towards a single cell catalyst: biodiesel (FAEE) production by various strains without exogenous ethanol supplementation.
Figure 5: Consolidated bioprocessing: growth and FAEE production by xylan-using strains.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Lynd, L. R., van Zyl, W. H., McBride, J. E. & Laser, M. Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, 577–583 (2005)

    Article  CAS  PubMed  Google Scholar 

  3. Hill, J., Nelson, E., Tilman, D., Polasky, S. & Tiffany, D. Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc. Natl Acad. Sci. USA 103, 11206–11210 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Magnuson, K., Jackowski, S., Rock, C. O. & Cronan, J. E. Regulation of fatty acid biosynthesis in Escherichia coli . Microbiol. Rev. 57, 522–542 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jiang, P. & Cronan, J. E. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Lu, X., Vora, H. & Khosla, C. Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab. Eng. 10, 333–339 (2008)

    Article  CAS  PubMed  Google Scholar 

  9. Dehesh, K., Jones, A., Knutzon, D. S. & Voelker, T. A. 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)

    Article  CAS  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Oil. Market Report, International Energy Agencyhttp://omrpublic.iea.org/omrarchive/16jan08full.pdf〉 (16 January 2008)

  13. Kalscheuer, R., Stolting, T. & Steinbuchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152, 2529–2536 (2006)

    Article  CAS  PubMed  Google Scholar 

  14. Rupilius, W. & Ahmad, S. The changing world of oleochemicals. Palm Oil Developments 44, 15–28 (2006)

    Google Scholar 

  15. Cheng, J. B. & Russell, D. W. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Metz, J. G., Pollard, M. R., Anderson, L., Hayes, T. R. & Lassner, M. W. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Ingram, L. O., Conway, T., Clark, D. P., Sewell, G. W. & Preston, J. F. Genetic engineering of ethanol production in Escherichia coli . Appl. Environ. Microbiol. 53, 2420–2425 (1987)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Adelsberger, H., Hertel, C., Glawischnig, E., Zverlov, V. V. & Schwarz, W. H. Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes. Microbiology 150, 2257–2266 (2004)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Qian, Z. G., Xia, X. X., Choi, J. H. & Lee, S. Y. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli . Biotechnol. Bioeng. 101, 587–601 (2008)

    Article  CAS  PubMed  Google Scholar 

  23. Tsuruta, H. 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)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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  PubMed  Google Scholar 

  25. Link, A. J., Phillips, D. & Church, G. M. 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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  27. Li, M. Z. & Elledge, S. J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 4, 251–256 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)

    Google Scholar 

  29. Aldai, N., Osoro, K., Barron, L. J. & Najera, A. I. 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)

    Article  CAS  PubMed  Google Scholar 

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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.

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

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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.

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Steen, E., Kang, Y., Bokinsky, G. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010). https://doi.org/10.1038/nature08721

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