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.

Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids

Nature Biotechnology volume 30, pages 354–359 (2012)Cite this article

Subjects

Abstract

Microbial production of chemicals is now an attractive alternative to chemical synthesis. Current efforts focus mainly on constructing pathways to produce different types of molecules1,2,3. However, there are few strategies for engineering regulatory components to improve product titers and conversion yields of heterologous pathways4. Here we developed a dynamic sensor-regulator system (DSRS) to produce fatty acid–based products in Escherichia coli, and demonstrated its use for biodiesel production. The DSRS uses a transcription factor that senses a key intermediate and dynamically regulates the expression of genes involved in biodiesel production. This DSRS substantially improved the stability of biodiesel-producing strains and increased the titer to 1.5 g/l and the yield threefold to 28% of the theoretical maximum. Given the large number of natural sensors available, this DSRS strategy can be extended to many other biosynthetic pathways to balance metabolism, thereby increasing product titers and conversion yields and stabilizing production hosts.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Design of FA/acyl-CoA biosensors.
Figure 2: Hybrid FA/acyl-CoA–regulated promoters.
Figure 3: Regulation of FAEE production by the DSRS.
Figure 4: Metabolite analysis and dynamic behavior of FAEE-producing strains.

References

  1. Ajikumar, P.K. et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330, 70–74 (2010).

    Article  CAS  Google Scholar 

  2. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Zhang, F. & Keasling, J.D. Biosensors and their applications in metabolic engineering. Trends Microbiol. 19, 323–329 (2011).

    Article  CAS  Google Scholar 

  5. Keasling, J.D. Manufacturing molecules through metabolic engineering. Science 330, 1355–1358 (2010).

    Article  CAS  Google Scholar 

  6. Harcum, S.W. & Bentley, W.E. Heat-shock and stringent responses have overlapping protease activity in Escherichia coli. Implications for heterologous protein yield. Appl. Biochem. Biotechnol. 80, 23–37 (1999).

    Article  CAS  Google Scholar 

  7. De Mey, M., Maertens, J., Lequeux, G.J., Soetaert, W.K. & Vandamme, E.J. Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering. BMC Biotechnol. 7, 34 (2007).

    Article  Google Scholar 

  8. Pfleger, B.F., Pitera, D.J., Smolke, C.D. & Keasling, J.D. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027–1032 (2006).

    Article  CAS  Google Scholar 

  9. Salis, H.M., Mirsky, E.A. & Voigt, C.A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

    Article  CAS  Google Scholar 

  10. Holtz, W.J. & Keasling, J.D. Engineering static and dynamic control of synthetic pathways. Cell 140, 19–23 (2010).

    Article  CAS  Google Scholar 

  11. Farmer, W.R. & Liao, J.C. Improving lycopene production in Escherichia coli by engineering metabolic control. Nat. Biotechnol. 18, 533–537 (2000).

    Article  CAS  Google Scholar 

  12. Zhang, F., Rodriguez, S. & Keasling, J.D. Metabolic engineering of microbial pathways for advanced biofuels production. Curr. Opin. Biotechnol. 775–783 (2011).

  13. Fujita, Y., Matsuoka, H. & Hirooka, K. Regulation of FA metabolism in bacteria. Mol. Microbiol. 66, 829–839 (2007).

    Article  CAS  Google Scholar 

  14. Cronan, J.E. Jr. In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor. J. Bacteriol. 179, 1819–1823 (1997).

    Article  CAS  Google Scholar 

  15. DiRusso, C.C., Heimert, T.L. & Metzger, A.K. Characterization of FadR, a global transcriptional regulator of FA metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A. J. Biol. Chem. 267, 8685–8691 (1992).

    CAS  PubMed  Google Scholar 

  16. Iram, S.H. & Cronan, J.E. Unexpected functional diversity among FadR FA transcriptional regulatory proteins. J. Biol. Chem. 280, 32148–32156 (2005).

    Article  CAS  Google Scholar 

  17. Henry, M.F. & Cronan, J.E. Jr. A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding. Cell 70, 671–679 (1992).

    Article  CAS  Google Scholar 

  18. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  Google Scholar 

  19. van Aalten, D.M., DiRusso, C.C. & Knudsen, J. The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR. EMBO J. 20, 2041–2050 (2001).

    Article  CAS  Google Scholar 

  20. Lanzer, M. & Bujard, H. Promoters largely determine the efficiency of repressor action. Proc. Natl. Acad. Sci. USA 85, 8973–8977 (1988).

    Article  CAS  Google Scholar 

  21. Spencer, A.K., Greenspan, A.D. & Cronan, J.E. Jr. Thioesterases I and II of Escherichia coli. Hydrolysis of native acyl-acyl carrier protein thioesters. J. Biol. Chem. 253, 5922–5926 (1978).

    CAS  PubMed  Google Scholar 

  22. Corchero, J.L. & Villaverde, A. Plasmid maintenance in Escherichia coli recombinant cultures is dramatically, steadily, and specifically influenced by features of the encoded proteins. Biotechnol. Bioeng. 58, 625–632 (1998).

    Article  CAS  Google Scholar 

  23. Friehs, K. Plasmid copy number and plasmid stability. Adv. Biochem. Eng. Biotechnol. 86, 47–82 (2004).

    CAS  PubMed  Google Scholar 

  24. Wilkinson, S.P. & Grove, A. Ligand-responsive transcriptional regulation by members of the MarR family of winged helix proteins. Curr. Issues Mol. Biol. 8, 51–62 (2006).

    PubMed  Google Scholar 

  25. Carothers, J.M., Goler, J.A., Juminaga, D. & Keasling, J.D. Model-driven engineering of RNA devices to quantitatively program gene expression. Science 334, 1716–1719 (2012).

    Article  Google Scholar 

  26. Lee, T.S. et al. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J. Biol. Eng. 5, 12 (2011).

    Article  CAS  Google Scholar 

  27. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).

    Article  Google Scholar 

  28. Hajimorad, M., Gray, P.R. & Keasling, J.D. A framework and model system to investigate linear system behavior in Escherichia coli. J. Biol. Eng. 5, 3 (2011).

    Article  CAS  Google Scholar 

  29. Ramsey, S., Orrell, D. & Bolouri, H. Dizzy: stochastic simulation of large-scale genetic regulatory networks. J. Bioinform. Comput. Biol. 3, 415–436 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank W. Holtz, E. Steen and N. Hillson for discussion and critical reading of the manuscript. This work was supported in part by the Synthetic Biology Engineering Research Center, which is funded by National Science Foundation award no. 0540879, and by the Joint BioEnergy Institute, which is funded by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231. F.Z. is supported by the Postdoctoral Fellowships Program of the Natural Sciences and Engineering Research Council of Canada.

Author information

Authors and Affiliations

  1. Joint BioEnergy Institute, Emeryville, California, USA

    Fuzhong Zhang & Jay D Keasling

  2. Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Fuzhong Zhang & Jay D Keasling

  3. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA

    Fuzhong Zhang & Jay D Keasling

  4. California Institute for Quantitative Biosciences, University of California, Berkeley, California, USA

    James M Carothers & Jay D Keasling

  5. Synthetic Biology Engineering Research Center, University of California, Berkeley, California, USA

    Jay D Keasling

  6. Department of Bioengineering, University of California, Berkeley, California, USA

    Jay D Keasling

Authors
  1. Fuzhong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James M Carothers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jay D Keasling
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.Z. and J.D.K. conceived the project and designed the experiments. F.Z. performed the experiments. J.M.C. designed and performed the DSRS modeling. F.Z., J.M.C. and J.D.K. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Jay D Keasling.

Ethics declarations

Competing interests

J.D.K. has a financial interest in Amyris, LS9 and Lygos.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–5 and Supplementary Figures 1–9 (PDF 860 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhang, F., Carothers, J. & Keasling, J. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 30, 354–359 (2012). https://doi.org/10.1038/nbt.2149

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2149

This article is cited by

Search

Advanced 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