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Engineering dynamic pathway regulation using stress-response promoters

Nature Biotechnology volume 31, pages 1039–1046 (2013)Cite this article

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Abstract

Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.

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Figure 1: Two modes of metabolic pathway regulation.
Figure 2: Systems biology analysis of FPP toxicity.
Figure 3: Production of amorphadiene using FPP-responsive promoters.
Figure 4: Fluorescence and amorphadiene production from inducible, constitutive and FPP-responsive promoters in EZ RDM with 1% glucose.
Figure 5: Targeted proteomic analysis of pathway enzymes (solid lines) and FPP accumulation profile (dashed lines) during the first 24 h of growth.

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Acknowledgements

The authors thank D. Pitera for the pADSmut plasmid and W. Holtz for the BglBrick plasmids with the lacUV5 promoter and LacIQ removed. J.A.-G. thanks Fundacion Ramon Areces for his PostDoc fellowship. This work was part of the Department of Energy Joint BioEnergy Institute (http://www.jbei.org/) supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.

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  1. Fuzhong Zhang

    Present address: Present address: Department of Energy, Environmental, and Chemical Engineering, Washington University, St. Louis, Missouri, USA.,

Authors and Affiliations

  1. Joint BioEnergy Institute, Emeryville, California, USA

    Robert H Dahl, Fuzhong Zhang, Jorge Alonso-Gutierrez, Edward Baidoo, Tanveer S Batth, Alyssa M Redding-Johanson, Christopher J Petzold, Aindrila Mukhopadhyay, Taek Soon Lee, Paul D Adams & Jay D Keasling

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

    Robert H Dahl, Fuzhong Zhang, Jorge Alonso-Gutierrez, Edward Baidoo, Tanveer S Batth, Alyssa M Redding-Johanson, Christopher J Petzold, Aindrila Mukhopadhyay, Taek Soon Lee, Paul D Adams & Jay D Keasling

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

    Robert H Dahl, Fuzhong Zhang & Jay D Keasling

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

    Paul D Adams & Jay D Keasling

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Contributions

R.H.D. and J.D.K. conceived the project. R.H.D. performed the growth experiments, microarrays and the construction of the responsive promoters. F.Z. constructed and analyzed the HMG-CoA biosensor. J.A.-G. analyzed the FPP sensor. E.B. performed metabolite analysis. T.S.B., A.M.R.-J. and C.J.P. performed the proteomic analysis. T.S.L., A.M. and P.D.A. directed aspects of the project. R.H.D., F.Z., J.A.-G. and J.D.K. wrote the manuscript.

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Correspondence to Jay D Keasling.

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

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Dahl, R., Zhang, F., Alonso-Gutierrez, J. et al. Engineering dynamic pathway regulation using stress-response promoters. Nat Biotechnol 31, 1039–1046 (2013). https://doi.org/10.1038/nbt.2689

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