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Chemical sensing of DNT by engineered olfactory yeast strain

Abstract

With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1α, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene–responsive receptor.

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Figure 1: Engineering of the WIF-1α strain.
Figure 2: GFP responses of WIF-1α–RI7 strain.
Figure 3: Shuttling of ligand-binding domains of other GPCRs in the WIF-1α system.
Figure 4: Screening of WIF-1α–OR strains for DNT-responsive colonies.

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GenBank/EMBL/DDBJ

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Acknowledgements

We would like to thank R. Reed (Johns Hopkins University School of Medicine), N. Gautam (Washington University School of Medicine) and J. Robishaw (Geisinger Clinic) for their kind gift of cDNA inserts encoding rat ACIII, β2 and γ5, respectively. Critical reading of the manuscript by J. Gardner, Z. Goldsmith and R. Saker is gratefully acknowledged. This work was sponsored by the US Defense Advanced Research Projects Agency through the Space and Naval Warfare Systems Center, San Diego, Contract No. N66001-00-C-8050.

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Contributions

V.R. carried out all the biochemical and fluorometric analyses. T.P.-C. made the receptor cassette and cloned Olfr226. M.J. cloned the receptors from rat olfactory epithelium. D.O. and J.H. assisted in the construction of the WIF-1 strain. D.D. conceived and supervised the project. All authors discussed the results, and D.D. wrote the manuscript.

Corresponding author

Correspondence to Danny N Dhanasekaran.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Amino acid sequences of rat Olfr226, mouse Olfr2 and mouse MOR226-1. (PDF 60 kb)

Supplementary Methods (PDF 101 kb)

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Radhika, V., Proikas-Cezanne, T., Jayaraman, M. et al. Chemical sensing of DNT by engineered olfactory yeast strain. Nat Chem Biol 3, 325–330 (2007). https://doi.org/10.1038/nchembio882

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