Programming olfaction into Pichia pastoris for high-throughput detection
of odors that influence mosquito behavior
Abstract
Mosquitoes utilize their sense of smell to locate prey and feed on their
blood. Repellents interfere with the biochemical cascades that detect
odors. Consequently, repellants are highly effective and
resource-efficient alternatives for controlling the spread of
mosquito-borne illnesses. Unfortunately, the discovery of repellents is
slow, laborious and error-prone. To this end, we have improved the speed
and accuracy of repellant discovery by constructing a whole-cell
biosensor for accurate detection of mosquito behavior-modifying
compounds such as repellants. We genetically engineered Pichia pastoris
to express the olfactory receptor co-receptor (Orco) of Anopheles
gambiae mosquitoes. This transmembrane protein behaves like a cationic
channel upon activation by stimulatory odorants. When the engineered
Pichia cells are cultured in calcium-containing Hank’s buffer, induction
of the medium with a stimulatory odorant results in influx of calcium
ions into the cells, and the stimulatory effect is quantifiable using
the calcium-sequestering fluorescent dye, fluo-4-acetoxymethyl ester.
Moreover, the stimulatory effect can be titrated by adjusting either the
concentration of calcium ions in the medium or level of induction of the
stimulatory odorant. Subsequent exposure of the activated Pichia cells
to a repellant molecule inhibits the stimulatory effect and quenches the
fluorescent signal, also in a titratable manner. Significantly, the
modular architecture of the biosensor allows easy and efficient
expansion of its detection range by co-expressing Orco with other
olfactory receptors. The high-throughput assay is also compatible with
robotic screening infrastructure, and our development represents a
paradigm change for the discovery of mosquito repellants.