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Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit

Nature Biotechnology volume 35, pages 273–279 (2017)Cite this article

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Abstract

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

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Figure 1: Characterization of a QS circuit to dynamically modulate a target gene of interest (GOI).
Figure 2: QS-based valve controlling Pfk-1 expression regulates cell growth and flux into central carbon metabolism.
Figure 3: Functionality of QS-based dynamic regulation in multiple culture media.
Figure 4: Glucaric acid production using the QS valve at the G6P branchpoint.
Figure 5: Shikimate production through the aromatic amino acid (AAA) biosynthesis pathway using the QS valve.

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Acknowledgements

We thank C. Collins at Rensselaer Polytechnic Institute (Troy, New York, USA) for providing plasmids and parts for the Esa QS system. In addition, we thank S. Arussy for help in preparing strains for glucaric acid production, and m2p-labs for the loan of a BioLector unit. This work was supported by the US National Science Foundation through the CAREER program (I.M.B.R. and K.L.J.P., Grant No. CBET-0954986), the Graduate Research Fellowship program (A.G.), the Synthetic Biology Engineering Research Center (SynBERC; A.G. and K.L.J.P., Grant No. EEC-0540879), and the Division of Molecular and Cellular Biosciences (A.G. and K.L.J.P., Grant No. MCB-1517913); by the Biotechnology Training Program of the National Institutes of Health (I.M.B.R., Grant No. T32GM008334); and by the USDA National Institute of Food and Agriculture Postdoctoral Fellowship (C.R.R., Grant No. 2013-67012-21022).

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Author notes

  1. Irene M Brockman Reizman & Christopher R Reisch

    Present address: Present addresses: Department of Chemical Engineering, Rose-Hulman Institute of Technology, Terre Haute, Indiana, USA (I.M.B.R.); Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA (C.R.R.).,

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Apoorv Gupta

  2. Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Apoorv Gupta & Kristala L J Prather

  3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Irene M Brockman Reizman, Christopher R Reisch & Kristala L J Prather

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Contributions

A.G., I.M.B.R., C.R.R. and K.L.J.P. designed and performed the experiments and analyzed the data. A.G., I.M.B.R. and K.L.J.P. wrote the manuscript.

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Correspondence to Kristala L J Prather.

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A.G., I.M.B.R. and K.L.J.P. are co-inventors on a patent application that includes the reported methods.

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1–3, Supplementary Figures 1–14, Supplementary Tables 1, 3, 4, and 5 (PDF 1705 kb)

Supplementary Table 2

All strains and corresponding genotypes relevant in this study (PDF 309 kb)

Supplementary Table 6

Promoter, 5′UTR and degradation tag sequences for the expression cassettes used in this study (PDF 71 kb)

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Gupta, A., Reizman, I., Reisch, C. et al. Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit. Nat Biotechnol 35, 273–279 (2017). https://doi.org/10.1038/nbt.3796

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