Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.