Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control

Abstract

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected M r of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 10 8 and 8.27 × 10 7 cells produce 229 and 114 µM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.

Eukaryotic algal community composition in tropical environments from solar salterns to the open sea

“…For Class IV, Tetraselmis had the most relatively abundant ASVs, a genus that is known to have many euryhaline species (Fon-Sing and Borowitzka, 2016), but has not been well studied in the tropics (Arora et al, 2013). The most relatively abundant Trebouxiophyceae ASVs were related to Picochlorum soloecismus and Chlorella desiccata, also known to be halotolerant, but not previously reported from tropical environments (Gonzalez-Esquer et al, 2019;Borovsky et al, 2020).…”

Trypsin modulating oostatic factor and its application as a larvicide for mosquito control: A review

“…Heat-killed Saccharomyces cerevisiae cells transformed with gene, tmfA and gfp-tmfA produced 95 percent mortality in A. aegypti (Borovsky et al 2018) [17] . Multiple insertions of the tmfA gene were performed in P. pastoris cells using a multi-copy integrating plasmid pICZB, resulting in greater expression of TMOF higher mortality of 88 percent within three days (Borovsky et al 2020) [18] .…”

“…Feeding A. aegypti larvae with P. pastoris and Escherichia coli expressing the tmfA and Bti toxin genes, respectively, revealed synergistic actions of TMOF and Cry proteins from Bti (Borovsky et al 2010) [13] . P. pastoris expressing the fusion protein of TMOF and Cry protein (cry4Aa-tmfA) caused about 90% mortality in A. aegypti larvae (Borovsky et al 2011) [19] .…”

Vector-Focused Approaches to Curb Malaria Transmission in the Brazilian Amazon: An Overview of Current and Future Challenges and Strategies

“…Genetic engineering methods have been proposed to increase the pathogenicity, improve longer-term efficacy, and prevent or delay insect resistance to entomopathogenic microorganisms [ 185 , 186 ]. The addition of Bti genes into Bs genomes to increase infectivity to mosquitoes [ 187 , 188 ] and the expression of Bti and non- Bti derived mosquito toxins in readily transformable microorganisms such as Chlorella desiccate , Pichia pastoris and Saccharomyces cerevisiae have been investigated as alternatives to develop new microbial products for mosquito control [ 189 , 190 , 191 ].…”