AUTHOR=Borovsky Dov , Nauwelaers Sabine , Shatters Robert 

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

JOURNAL=Frontiers in Physiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.00527

DOI=10.3389/fphys.2020.00527

ISSN=1664-042X

ABSTRACT=<p>Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of <italic>Aedes aegypti</italic>, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in <italic>Chlorella dessicata</italic> and in <italic>Saccharomyces cerevisiae</italic> cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, <italic>Pichia pastoris</italic>, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. <italic>P. pastoris</italic> was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: <italic>gfp-tmf</italic>A and <italic>tmf</italic>A inserted between <italic>Kpn</italic>I and <italic>Xba</italic>I in the multiple cloning site. The plasmid carries a strong AOXI promoter and <italic>P. pastoris</italic> 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 <italic>tmf</italic>A into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of <italic>tmfA.</italic> A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a <italic>tmfA</italic> transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with <italic>gfp-tmf</italic>A identified a protein band that ran at the expected <italic>M</italic><sub><italic>r</italic></sub> of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 10<sup>8</sup> and 8.27 × 10<sup>7</sup> 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 <italic>P. pastoris</italic> cells could be used in the future in the marsh to control mosquito populations.</p>