Team:Tokyo-NoKoGen

Overview

　Pesticides that are harmful to insects, such as cockroaches, put humans and the environment at risk, so safety pesticides are needed. Our project for iGEM 2014 is “Exterminator coli”. We engineered Escherichia coli (E.coli) that produces an inhibitor of a metabolic pathway for insects and exterminates insects such as roaches. In our project, we will produce a pesticide that has no effect on mammals and the environment using E.coli.

　Roaches and many other insects store their energy in the form of trehalose and use the enzyme trehalase to convert trehalose to glucose when needed. However, mammals store their energy in the form of glycogen, which does not require trehalase for conversion to glucose. Therefore trehalase inhibitor can be used as a pesticide which isn’t harmful to mammals but roaches.

　We engineered E.coli that produces the trehalase inhibitor, 3, 3'-diketotrehalose (3, 3'-dkT), a harmless pesticide to the environment and mammals. Our engineered E.coli will overexpress OtsA and OtsB which convert glucose-6-phosphate and UDP-glucose to trehalose, and glucose-3-dehydrogenase (G3DH) which converts trehalose to 3, 3'-dkT. Therefore, we predict that our E.coli can continue to produce 3, 3'-dkT by using glucose in insects. All we need to do is make roaches eat the E.coli we engineered !

Fig 1. Outline of our project, Exterminator coli.

Why did we focused on pesticide?

　Pesticides for insects are used almost everywhere -- not only in agriculture, but also in homes, parks, schools, buildings, forests, and roads. However, it is known that substances included in the pesticide have harmful influence for environment and also to humans. These harmful substances can be diffused in the environment. For example, organic phosphorus included in the agricultural chemicals affects nervous system, then cause miosis, vomiting, ataxia and muscular fasciculation. Also, pyrethroid included in spray pesticide does not cause serious poisoning unless it is overdose, but if we absorb surplus pyrethroid, it cause nausea, diarrhea, dizziness and convulsion. Furthermore, boric acid included in poison bait for cockroaches causes dehydration, gastroenteritis and lethargic sleep. There is a fatal case by overdose of boric acid. Therefore, we need pesticide that can exterminate only insects without any effect for environment and humans.

Mammal friendly pesticide -Trehalase inhibitor-

　Blood sugar of insects including cockroaches is trehalose which is one of the disaccharide. This means insects store trehalose as source of energy. Insects utilize trehalose in their cell metabolism by reconvering trehalose into glucose. Trehalose is converted to glucose by an enzyme called trehalase (α-glucoside-1-glucohydrolase) which hydrolyses trehalose. Then glucose will be phosphorylated, and used for synthesis of glycogen. Eventually, synthesized glycogen will be catabolized via glycolysis, or pentose phosphate pathway. Although mammals are unable to synthesis trehalose, they can use this sugar if it is present in their diet. In mammals, trehalase is restricted to microvilli membranes of the intestine and the kidney, so there is no indication of regulation of trehalase activity in mammals. Therefore inhibiting trehalase will not effect to mammals.

Fig 2.

　In our project, we focused on 3, 3’-diketotrehalose (3,3’-dkT) as trehalase inhibitor. The chemical structure of 3, 3’-dkT resembles trehalose, so the enzyme activity of trehalase will be prevented by competitive inhibition. Since it is reported that it can be synthesized via enzymatic reaction from trehalose, we assumed that we can make E. coli produce 3,3’-dkT and kill insects without harming mammals and environment.

Reference

Becker, A. et al ., The regulation of trehalose metabolism in insects, Experientia, (1996), Vol 52, Issue 5,

pp 433-439