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Using an AraC-based three-hybrid system to detect biocatalysts in vivo

Nature Biotechnology volume 18, pages 544–547 (2000)Cite this article

Abstract

Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.

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Figure 1: Overview of QUEST.
Figure 2: CID-mediated activation of the araBAD genes using strain MCFR as detected by MacConkey agar plates.
Figure 3: Detecting SD activity by MacConkey agar plates.
Figure 4: Detecting SD activity by fluorescence.
Figure 5: Sensitivity of QUEST.

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Acknowledgements

The authors wish to acknowledge the kind gift of the DDBO, plasmids, and strains used in this work (D. Jordan, G. Basarab, R. Schlief, J. Steffans, and M. Casadaban). The authors also thank M. Ostermeier for a critical reading of the manuscript. S.M.F. is funded by a fellowship from the Damon Runyon-Walter Winchell Foundation and F.S. is funded by a fellowship from the National Institutes of Health.

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  1. Andrew E Nixon

    Present address: Dyax Corporation, One Kendall Square, Building 600, 5th Floor, Cambridge, MA, 02139

Authors and Affiliations

  1. Department of Chemistry, The Pennsylvania State University, University Park, PA 16802

    Steven M Firestine, Frank Salinas, Stephen J Baker & Stephen J Benkovic

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  1. Steven M Firestine
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Correspondence to Stephen J Benkovic.

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Firestine, S., Salinas, F., Nixon, A. et al. Using an AraC-based three-hybrid system to detect biocatalysts in vivo. Nat Biotechnol 18, 544–547 (2000). https://doi.org/10.1038/75414

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