Team:Cologne-Duesseldorf/Applied Design

Applied Design

Nootkatone – a promising alternative for insect repellents

Imagine: It’s a hot summer evening and you are sitting in the garden with your friends, having a good time! Rather a beautiful thought. If only it wasn’t for all these annoying insects like mosquitoes which are getting on your nerves. Of course having an insect bite is not comfortable but all in all the bite will itch for 3 days and then it’s over.

In Central America a mother tucks in her little son and kisses him goodnight. Under the cover of darkness a black, barely 3 cm long assassin bug crawls out of a gap of the ceiling and falls unnoticed on the face of the little boy. The bug drills his proboscis into the soft skin. While the insect is sucking the blood, it defecates on the skin. Unsuspectingly the little boy scratches his face and rubs the infected feces under his skin. Within one to two weeks the boy develops high fever and his body starts to swell up. He is infected with the Chagas disease. Even if he survives the fever the parasite will start attacking the heart, nerves and the internal tissue.

Eventually time will pass, possibly without any symptoms. But after about ten or more years pathological changes in the digestive tract and symptoms of encephalitis might appear finally resulting in heart failure.
Even though this story is purely fictional, it describes the fate of many people who might get infected.
Chagas is one example but there are so much more dangerous diseases like malaria, Zika, dengue caused by insects.

Different agents are used for the protection against insects, the most effective ones being Diethyltoluamid (DEET), Icaridin and Para-Menthan-3,8-Diol. The world‘s most commonly used insect repellent is DEET. Scientists long surmised that DEET works by blocking insect olfactory receptors for 1-octen-3-ol, a volatile substance that is contained in human sweat and breath [1]. However recent studies show that insects are able to detect DEET and other smells with olfactory and gustatory receptors which means they can sense DEET and avoid it [2]. DEET is often sold and used in spray or lotion with varying concentrations up to 100%. A concentration of 75% provided more than 90% reduction of mosquito bites for up to 8h post-treatment [3]. Even though these are really promising results DEET has side effects. It can act as an irritant and cause epidermal reactions, breathing difficulty, burning eyes and headaches [4]. One really terrifying fact is that in the DEET Reregistration Eligibility Decision (RED) in 1998, the United States Environmental Protection Agency (EPA) reported 14 to 46 cases of potential DEET-associated seizures, including 4 deaths [5]. Another, meanwhile in Europe and USA forbidden insect repellent, is Dichlordiphenyltrichlorethan (DDT). DDT was the world’s most commonly used insect repellent until the 1970s. It was used in the second half of World War II to control malaria and typhus among the troops and the civilians. For his discovery of the high efficiency of DDT as a contact poison against several arthropods the scientist Paul Hermann Müller was awarded the Nobel Prize in Physiology or Medicine in 1948. Because of its effectiveness it was promoted by government and industry for use as an agricultural and household pesticide. At this point of time nobody foresaw the immense consequences the usage of DTT could have, except Rachel Carson. She had concerns about its use and observed the environmental impacts that coincided with widespread use of DDT in agriculture in the United States and published the book Silent Spring. The book claimed that DDT can cause cancer and the usage in agriculture was a threat to wildlife, in particular for birds. Its publication was a seminal event for the environmental movement and resulted in a large public outcry that eventually led to a ban on DDT's agricultural use in the United States in 1972. A worldwide ban on agricultural use was formalized under the Stockholm Convention on Persistent Organic Pollutants. In India the use of DDT in order to fight malaria and leishmaniasis is still allowed due to its high effectiveness [6].

To conclude: Until now there is no effective insect repellent without any side effects for public sale! This situation is absolutely inacceptable and searching for an alternative is of high importance.

As we don’t want to be sitting around but do something we discovered an effective alternative – the sesquiterpene nootkatone. Nootkatone is a flavoring substance which is present in the peel of grapefruits and is responsible for its characteristic scent. It has been proven that nootkatone is toxic to the disease-vectoring insects [7]. It is nontoxic to humans and commonly used in foods, cosmetics, and pharmaceuticals [8]. Furthermore since it is a natural ingredient in some foods it is officially classified as safe [9]. Additionally, it is environmentally friendly, volatile and does not persist in the environment. Right now, nootkatone costs $4000 per kilogram. This makes it impossible to use it as an insect repellent as it’s not affordable, especially for poor countries where the need for repellents is immense. There are different production ways but all have high costs. The isolation from nootkatone from natural plant sources suffers from low yields, and chemical synthesis involves carcinogenic or hazardous compounds. Many of the production pathways are based on the bicyclic sesquiterpene (+) valencene which is the main component of the volatile oil from the Valencia-Orange and grapefruits. Biotechnological approaches to produce nootkatone in fungi, bacteria and plants all have one major problem in common. The pathway of nootkatone is toxic for the cells [10].

Here is where our compartmentation comes into play. We want to implement the whole pathway into our modified peroxisome in yeast. Peroxisomes are known for accommodating toxic pathways so this compartment fits our needs just perfectly.
This implementation of the pathway may lead to a high and cheap production of nootkatone.

[1] Science 319 (5871): 1838–1842, Insect Odorant Receptors Are Molecular Targets of the Insect Repellent DEET

[2] Nature 478, 511–514 (27. Oktober 2011), A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor

[3] Journal of the American Mosquito Control Association. 12 (1): 69–74 (1996), Essential oil analysis and field evaluation of the citrosa plant Pelargonium citrosum as a repellent against populations of Aedes mosquitoes

[4] "Insect Repellent Use and Safety". West Nile Virus. Centers for Disease Control and Prevention. 2007-01-12

[5] "Reregistration Eligibility Decision: DEET", U.S. Environmental Protection Agency, Office of Prevention, Pesticides, and Toxic Substances. September 1998. pp. 39–40

[6] Current Science 85, 11, 1532–153, 2003, V.P. Sharma: DDT: The fallen angel

[7] Journal of Chemical Ecology (March 2001), Volume 27, Issue 3, pp 523–531, Nootkatone Is a Repellent for Formosan Subterranean Termite (Coptotermes formosanus)

[8] USDA (January 2011), "Lignin + Nootkatone = Dead Ticks", Jan Suszkiw

[9] NPR, Richard Knox (April 18, 2011). "Repelling Bugs With The Essence Of Grapefruit"

[10] Metabolic Engineering Volume 18, July 2013, Pages 25-35, Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae