Team:IISER-Mohali/History
Background
Proof of concept that cat pheromone repels rats
Cat urine has been reported to contain pheromone precursors and major urinary proteins that the mice brains are biologically programmed to perceive as a threat, scurrying them away at the very slightest detection. Using synthetic biology, our team has reconstructed the cat felinine biosynthetic pathway in Saccharomyces cerevisiae.The proof of concept is based on results obtained from Papes, Logan and Stowers, 2010.
This study has verified that a predator’s odor is a sufficient condition for inducing stereotyped fear responses in rodents, which occur as three defensive behaviors - avoidance, risk assessment, and increased ACTH levels. This also verified that the fear response is an innate mechanism, not a learned behaviour, since the mice tested were raised for generations without encountering a predator’s odor. On fractionating the odor responsible for fear responses in rodents, it was hinted that a class of proteins called major urinary proteins (MUPs) were sufficient. Purified recombinant protein expression studies confirmed these findings.
Felinine Pathway in cats
Domestic cats spray urine containing species-specific odor for territorial marking. Felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid), a putative pheromone precursor, is excreted in cat urine. Most mammals have a highly developed olfactory sense. Urinary odorants and pheromones are crucial for chemical communication used in reproduction, territoriality, and conspecific recognition. (Nat. Rev. Neurosci., 4 (2003), pp. 551-562)
A felinine precursor is present in cat blood as 3-methylbutanol-glutathione (3-MBG) and is formed via a glutathione conjugation reaction between glutathione (GSH) and isopentenyl pyrophosphate (IPP) as an intermediate of cholesterol biosynthesis (J. Biol. Chem., 277 (2002), pp. 114-119). In general, glutathione S-conjugates such as 3-MBG are converted to cysteinylglycine S-conjugates (3-MBCG) by γ-glutamyl transferase in the kidney and the cysteinylglycine S-conjugates are hydrolyzed by cauxin to give rise to felinine, the precurser of cat pheromone.
Proposed Pathway for Felinine synthesis in Saccharomyces cerevisiae
We know that GSH and IPP conjugates to give 3-MBG in cat (Felis catus) and both these substrates are already present in the cytoplasm of Saccharomyces cerevisiae. Through bioinformatics analysis and literature mining, we found that the cat homologs of γ-GGT and cauxin are also being produced in Saccharomyces cerevisiae (Mehdi, K., Thierie, J., & Penninckx, M. J. (2001)). Although the cauxin and γ-GGT homologs are found in Saccharomyces cerevisiae, they are localized in the vacuoles and perform a similar function. Therefore, we hypothesize that they will be available to act on the respective substrates and produce felinine only in vacuole. Fortunately, to aid with the same, there also exists a Glutathione conjugated pump called YCF1 found in the vacuolar membrane of Saccharomyces cerevisiae. This pump plays a crucial role in detoxification and transports glutathione conjugates that are not immediately reduced in cytosol to vacuole. Therefore the putative GST needed for the conjugation must be introduced in the cytoplasm, which can be further metabolized through the above mentioned pathway after entering the vacuole through the YCF1 pump. These properties of Saccharomyces cerevisiae coupled with the fact that their cells use pheromone based intracellular signal transduction pathway for mating made us choose Saccharomyces cerevisiae as our model organism for the production of cat pheromone.
The figures above show the proposed schematic pathway for felinine synthesis in Saccharomyces cerevisiae.
Therefore our next course of action will be towards finding or introducing a GST homolog in Saccharomyces cerevisiae. This involves bioinformatic analysis to identify Saccharomyces cerevisiae homologs of the genes involved in the cat felinine biosynthetic pathway and cloning of the required gene in order to express it in Saccharomyces cerevisiae. Since Saccharomyces cerevisiae has isoprenol in addition to isoprenol pyrophosphate, we predict that isoprenol-GSH conjugation can also exist. This conjugation will result in products other than felinine being formed in Saccharomyces cerevisiae.
