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Title: Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a co-product in an ethanologenix Saccharomyces cerevisiae strain

Author
item Hughes, Stephen
item Dowd, Patrick
item Hector, Ronald - Ron
item PANAVAS, TADAS - LIFESENSORS
item STERNER, DAVID - PROGENRA INC
item Qureshi, Nasib
item Bischoff, Kenneth
item BANG, SOOKIE - SDSM&T
item Mertens, Jeffrey
item Johnson, Eric
item Li, Xin Liang
item Jackson Jr, John
item CAUGHEY, ROBERT - UNIV OF IL
item RIEDMULLER, STEVEN - HUDSON CONTROL
item BARTOLETT, SCOTT - HUDSON CONTROL
item Liu, Siqing
item Rich, Joseph
item FARRELLY, PHILIP - HUDSON CONTROL
item BUTT, TAUSEEF - LIFESENSORS
item LABAER, JOSHUA - HARVARD INSTITUTE
item Cotta, Michael

Submitted to: Journal of Peptide Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2008
Publication Date: 3/9/2008
Citation: Hughes, S.R., Dowd, P.F., Hector, R.E., Panavas, T., Sterner, D.E., Qureshi, N., Bischoff, K.M., Bang, S.B., Mertens, J.A., Johnson, E.T., Li, X., Jackson Jr, J.S., Caughey, R.J., Riedmuller, S.B., Bartolett, S., Liu, S., Rich, J.O., Farrelly, P.J., Butt, T.R., Labaer, J., Cotta, M.A. 2008. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a co-product in an ethanologenix Saccharomyces cerevisiae strain. Journal of Peptide Science. 14(9):1039-1050. Available: http://www3.interscience.wiley.com/cgi-bin/fulltext/119030240/PDFSTART.

Interpretive Summary: New biological pest control methods are needed because insects are developing resistance to current pesticides. A peptide toxin from spider venom was improved by mutation and expressed in yeast cells using a novel, highly efficient vector system. The toxin killed fall armyworms, a major cause of damage to corn. The yeast is a strain used for industrial fuel ethanol production and the pesticide may be expressed as a valuable co-product in the process.

Technical Abstract: 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 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 open reading frames were also carrying the xylose isomerase 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 a-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 co-products to increase the cost-effectiveness of fuel ethanol bioproduction.