PHEROMONES

DMDS DMTS

One part of our project is based on the use of pheromones. Some studies have shown that dmds (dimethyl disulfide) and dmts (dimethyl trisulfide) are the most attractive pheromones for bed bugs [1]. Otherwise, those molecules are present in bed bugs dejections, which allows them to find back their colonies when they go outside in the night. In order to make the bed bugs coming into the trap, we want to creat an enginered E. coli, that will produce those 2 pheromones and stay in the trap.

Benzyl alcohol

The project goal is to eliminate bed bugs by using enginered E. coli bacteria that can produce DMDS and DMTS pheromones, and by creating a contact between Beauveria Bassiana and the bed bug that will come into the trap. It will come back then to its colony and infect other bed bugs with our fungus when it will die. This infection will hapenned by fungus sporulation, and also by the contact with the infected cuticule. In order to attract healthy bed bugs to the dead body, we want the infected bed bug to going out of our trap with an E. coli enginered strain on its cuticule (as the fungus), that could also produce pheromones and stay hooked on the insect. The problem is that some studies have shown that DMDS and DMTS have growth inhibitors effect on B. bassiana. That is why we’ve decided to creat an other enginered E.coli strain, that could produce benzyl alcohol. Benzyl alcohol is also a pheromone that can attract bed bugs To do so, we’ve decided to insert a no-natural pathway in an E. coli strain, that derived of an other natural pathway, which is the glucose degradation in phenylalanine. This process aims to use the phenylpyruvate (one of this natural pathway’s substrats) in order to start the benzyl alcohol biosynthesis pathway. It needs 3 differents enzymes: 4-hydroxyphenylpyruvate dioxygenase, (S)-mandelate dehydrogenase and benzoylformate decarboxylase, which are respectivly encoded by hmaS, mdlB and mdlC genes. We’ve worked on those 3 differents genes, coming from different organism : Pseudomonas fluorescens (mdlB and mdlC) and Amycolatopsis orientalis (hmaS), because of there similitudes with E.coli. Those genes will be united as an operon with an RBS and strong promoteur construction, and that operon will be insert into a plasmid that will be introduced into an E.coli enginered strain.

hmaS 4-hydroxyphenylpyruvate synthase, or 4-hydroxyphenylpyruvate dioxygenase 2 is an enzyme that catalyzes the chemical reaction bellow, by acting as a decarboxylase and a hydroxylase: 4-hydroxyphenylpyruvic acid + O2 <=> 4-hydroxymandelate+ CO2. It belongs to oxidoreductases family and is involved in the vancomycin biosynthesis pathway. This enzyme is expressed by hmaS gene, that is present in Amycolatopsis orientalis. It is the first benzyl alcohol pathway step for its biosynthesis.