Insecticidal activity of recombinant avidin produced in yeast Gareth Hinchliffe a , David P. Bown a , John A. Gatehouse a , Elaine Fitches b, * a School of Biological and Biomedical Sciences, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK b Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, UK 1. Introduction Avidin, a protein found in the egg white of birds, reptiles and amphibians, is thought to function as an antibacterial, host-defence protein, by virtue of its ability to bind biotin, an essential vitamin for most organisms. In nature, avidin exists as a homotetramer, comprising four 16 kDa singly glycosylated subunits, each able to bind a biotin molecule with extreme specificity and affinity (Green, 1990). The search for protein-based approaches to crop protection has been prompted by the growing demand for more environmentally compatible approaches to pest control, and encouraged by the successful use of insecticidal protein toxins from the soil microor- ganism Bacillus thuringensis (Bt) as a spray and in transgenic crops. The insecticidal activity of avidin was first identified 50 years ago, when dietary avidin was found to have detrimental effects on the growth of housefly larvae (Musca vicina)(Levinson and Bergmann, 1959). Avidin, and the related bacterial protein streptavidin, have since been shown to be toxic to a wide range of insects including representatives of Lepidoptera (Morgan et al., 1993; Du and Nickerson, 1995; Markwick et al., 2001; Burgess et al., 2002; Zhu et al., 2005), Diptera (Levinson and Bergmann, 1959; Tsiropoulos, 1985; Bruins et al., 1991), Coleoptera (Levinson et al., 1967; Allsopp and McGhie, 1996; Kramer et al., 2000; Yoza et al., 2005; Cooper et al., 2006; Murdock and Shade, 2008) and Orthoptera (Christeller et al., 2000). Avidin is a normal component of human diet, and thus has potential for adoption as an insect control agent. Furthermore, dietary levels of the protein required for effective insecticidal activity against a wide range of pests are much lower than levels present in normal human diet. Expression of avidin in transgenic plants has been put forward as a strategy to confer protection against insect pests, with engineering of maize, corn, tobacco, potato, apple and rice having been reported (Kramer et al., 2000; Burgess et al., 2002; Murray et al., 2002; Markwick et al., 2003; Yoza et al., 2005). However, the initial report of expression of avidin in maize, with the intention of producing protein which could be extracted for use as a biochemical reagent (Hood et al., 1997) remains the only example of large-scale expression in planta. The insecticidal effects of avidin are mediated through its biotin- binding activity. In many cases insecticidal effects have been shown to be eliminated in diets supplemented with biotin, supporting the hypothesis that avidin acts through the sequestration of biotin from ingested food, thereby preventing absorption and causing biotin deficiency in the insect. Biotin, as a cofactor of major carboxylases involved in key process such as gluconeogenesis, lipogenesis, and fatty acid and amino acid catabolism is essential for insect growth (Wood and Barden, 1977; Knowles, 1989). Most insects fed on avidin-containing diets show retarded growth, leading to eventual Journal of Insect Physiology 56 (2010) 629–639 ARTICLE INFO Article history: Received 22 July 2009 Received in revised form 22 January 2010 Accepted 24 January 2010 Keywords: Insecticidal protein Biotin binding Yeast expression system Hemiptera ABSTRACT An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 mM and 15 mM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations 3.8 mM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC 50 of 2.1 mM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 mM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin. Crown Copyright ß 2010 Published by Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +44 01904 462564; fax: +44 01904 4622111. E-mail address: elaine.fitches@fera.gsi.gov.uk (E. Fitches). Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 0022-1910/$ – see front matter . Crown Copyright ß 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.01.007