Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes

Authors: BOROVSKY, DOV - OAK RIDGE INSTITUTE FOR SCIENCE AND EDUCATION (ORISE); NAUEWELAERS, S - UNIVERSITY OF FLORIDA; POWELL, CHARLES - UNIVERSITY OF FLORIDA; Shatters, Robert - Bob

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2017
Publication Date: 1/18/2017
Citation: Borovsky, D., Nauewelaers, S., Powell, C., Shatters, R.G. 2017. Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes. Journal of Insect Physiology. 106(2):134-146. https://doi.org/10.1016/j.jinsphys.01.008.
DOI: https://doi.org/10.1016/j.jinsphys.01.008

Interpretive Summary: Trypsin modulating oostatic factor is a peptide hormone isolated from mosquitoes that controls blood meal digestion and egg development in female mosquitoes. It was previously shown that this peptide is toxic to mosquito larvae and other agricultural pest insects when fed orally in diets or even when produced in transgenic plants on which the pest insect feeds. In this work it was demonstrated that this peptide could be produced in yeast and these yeast cells could be harvested and fed to pest insects (i.e. mosquitoes) as a way of killing the pest insect. This work has demonstrates the potential for the use of this yeast expression system in producing a topically applied insect specific toxin that could be used to control various pest insects.

Technical Abstract: Trypsin modulating oostatic factor (TMOF), a decapaptide isolated from the ovaries of Aedes aegypti, is the physiological factor that terminates the trypsin biosynthesis after the blood meal. Earlier results obtained from feeding mosquito larvae and injecting female mosquitoes with TMOF show that trypsin biosynthesis and egg development are inhibited, indicating that TMOF traverses the gut epithelial cells and modulates trypsin biosynthesis, making it a potential larvacidal peptide hormone. Therefore, TMOF and TMOF green fluorescent protein (GFP) fusion protein with a trypsin cleavage site,allowing TMOF release in the larval gut, were expressed in S. cerevisiae cells that were transformed using homologous recombination at ura3-52 with an engineered plasmid (pYDB2) carrying tmfA and gfp-tmfA and a strong galactose promoter (PGAL1). Southern blot analyses showed that each cell incorporated a single tmfA or gfp-tmfA. Western blot analyses of cells that were fermented up to 48 h showed that the engineered S. cerevisiae cells synthesized both TMOF and GFP-TMOF and heat treatment did not affect the recombinant proteins. Engineered S. cerevisiae (3 108 cells) that were fermented for 4 h produced (2.1 ± 0.2 lg ± S.E.M) of TMOF. Feeding the engineered cells producing TMOF and GFP-TMOF to larval mosquito caused high mortalities (66 ± 12% and 83 ± 8%, respectively). S. cerevisiae cells transfected with pYEX-BX carrying gfp-tmfA and (DPAR)4 or transformed by homologous recombination of pYDB2-gfptmfA carrying a heat shock promoter (PHP) were ineffective. Engineered heat treated yeast cells are consumed by mosquito larvae, and could be used to control mosquitoes.