Team:Tokyo-NoKoGen

Overview

　Pesticides are not only harmful to insects, such as cockroaches, but also pose a risk to humans and the environment. A safe pesticide is greatly needed. Our project for iGEM 2014 is “Exterminator coli”, an Escherichia coli machine that exterminates insects such as roaches while being completely safe to mammals and the environment.

　Roaches and many other insects store their energy in the form of trehalose, which can be converted to glucose, when needed, by the enzyme trehalase. However, mammals store their energy in the form of glycogen, which does not require trehalase for conversion to glucose. Trehalase inhibitors can therefore be used as a pesticide that can control roaches while being harmless for mammals and the environment.

　We engineered E. coli that produces the trehalase inhibitor, 3,3´-diketotrehalose (3,3´-dkT), a pesticide that is harmless for the environment and mammals. Our engineered E. coli will overexpress trehalose 6-phosphate synthase (OtsA) and trehalose 6-phosphate phosphatase (OtsB), which convert glucose-6-phosphate and UDP-glucose to trehalose, and glucose-3-dehydrogenase (G3DH), which converts trehalose to 3,3’-dkT. We predict that Exterminator coli will continue to produce 3,3’-dkT even after it is ingested by the insect.

Fig. 1 Outline of our project, Exterminator coli.

Why did we focus 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, organophosphates included in the agricultural chemicals affects nervous system, then cause miosis, vomiting, ataxia and muscular twitching. 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-

　Many insects, including cockroaches, store their energy in the form of the disaccharide trehalose in their blood. Insects utilize trehalose in their cell metabolism by reconverting it into glucose by the enzyme trehalase (α-glucoside-1-glucohydrolase), which hydrolyses trehalose (1). The glucose can then be phosphorylated and used for glycogen synthesis or catabolized via glycolysis or the pentose phosphate pathway. Although mammals do not synthesize trehalose, they possess a trehalase in their intestine and kidney, which allows them assimilate trehalose if present in their diet. A trehalose inhibitor will therefore not pose any significant harm to mammals.

Fig. 2 Synthesis of trehalose in insects

　We discovered from the literature that 3,3’-diketotrehalose (3,3’-dkT) can be used as a trehalase inhibitor (Kojima et al, 2001). Trehalose is converted to 3,3’-dkT by the enzymatic reaction of glucose 3 dehydrogenase (G3DH). The chemical structure of 3,3’-dkT resembles trehalose, so the enzyme activity of trehalase will be prevented by competitive inhibition.

Reference

(1) Becker, A. et al . (1996) The regulation of trehalose metabolism in insects. Experientia , 52, 433-439