Team:Queens Canada/Nootkatone
Nootkatone
Overview
Our project focused on the diagnosis of Lyme disease in ticks, however, it is equally important to prevent this disease. Ideally, the two best ways to prevent Lyme disease would be A) a vaccine against Lyme disease or B) a bug spray the prevents tick bites. In the latter case, a bug spray would also prevent other tick-borne diseases and would be more universally applicable as well as affordable.
At the moment, there are many bug sprays on the market that ticks respond very well to, namely those with active ingredients such as DEET, IR3535, Picaridin, PMD, and 2-undecanone according to the center of disease control (CDC) (1). The problem with most of the active ingredients in these is that they create harmful runoff products that have the potential to damage the ecosystem. Many of these chemicals, namely DEET, have had adverse effects reported such as neurotoxicity in high quantities as well as can have adverse effects on off-target organisms. Additionally, often, these products must be chemically synthesized which can be cost-inefficient.
Our solution is nootkatone. Nootkatone is the chemical that gives grapefruits their unique citrusy smell and also is present in minute quantities in Alaskan Yellow Cedar (Cupressus nootkatensis) trees to repel termites and mold (2). Nootkatone is a naturally occurring chemical that has no known adverse effects on humans, animals, birds, plants, and the environment and effectively repels ticks.
The problem is that nootkatone is very difficult to synthesize in vitro or in a pure chemistry sense as there are many stereoisomers that arise in the synthesis process, only one of which is useful against ticks (3). Furthermore, the synthesis of nootkatone chemically uses many harmful reagents. It has been suggested that intermediates from the metabolic pathways of Saccharomyces cerevisiae can be taken advantage of and converted to nootkatone via two additional steps (4). These additional steps consist of a step that takes farnesyl pyrophosphate from yeast’s prenoid biosynthesis pathway and converts it into (+)-Valencene via the enzyme Valencene Synthase, derived from Cupressus nootkatensis (5, 6). (+)-Valencene can then be converted to (+)-Nootkatone via Valencene Oxidase also derived from Cupressus nootkatensis (6). By engineering a bifunctional protein from these two enzymes and engineering them into Saccharomyces cerevisiae cells, nootkatone can be synthesized in a safe and environmentally friendly way.
References
1. Aliea Hruetic (2021) How to Prevent Tick Bites. [online] https://www.prevention.com/health/a22095155/best-tick-repellents/ (Accessed July 12, 2021)
2. Nootkatone: A new active ingredient for developing insecticides and insect repellents | Division of Vector-Borne Diseases | NCEZID | CDC (2020) Centers Dis. Control Prev. Natl. Cent. Emerg. Zoonotic Infect. Dis. (NCEZID), Div. Vector-Borne Dis. [online] https://www.cdc.gov/ncezid/dvbd/media/dpk-nootkatone.html (Accessed July 12, 2021)
3. Bing Hong, Raphaël Lebeuf, Stéphanie Delbaere, Paul L. Alsters, V. N.-R. (2016) One-Pot Synthesis of (+)-Nootkatone via Dark SingletOxygenation of Valencene: The Triple Role ofthe Amphiphilic Molybdate Catalyst. Catalysts
4. Meng, X., Liu, H., Xu, W., Zhang, W., Wang, Z., and Liu, W. (2020) Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid (+)-nootkatone. Microb. Cell Fact. 10.1186/S12934-020-1295-6
5. Beekwilder, J., Houwelingen, A. van, Cankar, K., Dijk, A. D. J. van, Jong, R. M. de, Stoopen, G., Bouwmeester, H., Achkar, J., Sonke, T., and Bosch, D. (2014) Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene. Plant Biotechnol. J. 12, 174–182
6. Cankar, K., Houwelingen, A. van, Goedbloed, M., Renirie, R., Jong, R. M. de, Bouwmeester, H., Bosch, D., Sonke, T., and Beekwilder, J. (2014) Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis). FEBS Lett. 588, 1001–1007