Expression of spider toxin in entomopathogenic fungus Lecanicillium muscarium and selection of the strain showing efficient secretion of the recombinant protein

Abstract

ABSTRACT Beta/delta-agatoxin-1 of spider Agelena orientalis was expressed in entomopathogenic fungus Lecanicillium muscarium. To ensure secretion of the recombinant product by the fungus, the signal secretory peptide of the Metarhizium anisopliae Mcl1 protein was inserted into the sequence. For detection of the recombinant product and selection of transformants, the toxin sequence was also fused with eGFP at the C-terminus. The gene encoding the A. orientalis toxin with the Mcl1 protein signal peptide was commercially synthesized, amplified and cloned into the vector pBARGPE1 designed for heterologous expression under the control of the PgpdA promoter and the trpC terminator of Aspergillus nidulans. A double selection on selective medium and microscopic analysis of transformants allowed obtaining a mitotically stable recombinant strain of L. muscarium. The recognition of the Mcl1 derived signal peptide in the cells of transformants and effective secretion of the hybrid product was confirmed by immunoblotting.