Vol.:(0123456789) 1 3 Current Microbiology https://doi.org/10.1007/s00284-018-1578-x Tyrosine-776 of Vip3Aa64 from Bacillus thuringiensis is Important for Retained Larvicidal Activity During High-Temperature Storage Sumarin Soonsanga 1  · Amporn Rungrod 1  · Mongkon Audtho 1  · Boonhiang Promdonkoy 1 Received: 15 May 2018 / Accepted: 3 October 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Vip3Aa (vegetative insecticidal protein) secreted by Bacillus thuringiensis (Bt) is highly toxic to lepidopteran insects. The Bt isolate M190 produces Vip3Aa35 at high concentrations, and Vip3Aa35 was found to be very efective against Spodoptera exigua. Unfortunately, the use of Vip3Aa35 in pest control is limited by its short shelf life when stored at high temperatures, retaining activity for only 1 month at 37 °C. To fnd a more stable alternative, we screened 500 isolates of Bt collected from various locations in Thailand and discovered Bt isolate 294 which produced large amounts of Vip3Aa64 that exhibited high toxicity against S. exigua but could be stored at 37 °C for up to 3 months. Vip3Aa35 and Vip3Aa64 have only nine amino acid diferences between them, with six of those residues being located at the C terminus. Vip3Aa35 and Vip3Aa64 chime- ras revealed that the C-terminal sequence is important for the retained larvicidal activity observed with Vip3Aa64. Various single amino acid substitutions were created to identify the key amino acids responsible for this stability. A single residue, Tyr776, was found to be solely responsible, with the Vip3Aa35:N776Y acquiring thermostability similar to Vip3Aa64 while the Vip3Aa64:Y776N exhibited Vip3Aa35-like thermostability. Introduction Bacillus thuringiensis (Bt) is widely used as a biocontrol agent to control insect pests due to the toxicity of crystal proteins (Cry) produced during sporulation. However, stud- ies indicate that Vip3A (vegetative insecticidal protein) also contributes to the killing of lepidopteran insect larvae [1]. Its mode of action difers from that of Cry proteins [2], and therefore vip3A genes have been expressed in cotton and corn together with Cry genes to provide higher-level protec- tion and delay insect resistance [3–5]. In addition, Vip3A was evaluated to be safe for use as a biopesticide since recombinant Bt Vip3A was harmless to rodents at dose of 5000 mg/kg bodyweight per day [6], and transgenic maize MIR162 expressing Vip3A did not pose any risk to non- target organisms [7]. Vip3A is produced during the vegetative growth phase and is secreted into the culture medium. Secreted Vip3A is about 88 kDa in size, but is proteolytically processed into an approximately 62–65 kDa active form (amino acid 199 to the end of Vip3A) by larvae gut juice [1]. This fragment binds to specifc receptors and lyses midgut epithelium cells, leading to larvae death [8] through as-yet unidentifed mechanisms. Structural information of Vip3A is currently unavailable because its sequence shares no homology with other known proteins and protein crystallization has proven to be very difcult [9]. A common strategy for Vip3A application in insect pest control is to express the gene in transgenic plants together with other insecticide genes. There is no commercial product of the protein available for direct application due to high production costs and protein instability. This presents a great barrier for its application in countries with restrictions on the use of genetically modifed organisms (GMO). Therefore, solving the issue of protein instability would immensely facilitate direct application of Vip3A, especially since its short half-life at high temperatures complicates application in feld conditions. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00284-018-1578-x) contains supplementary material, which is available to authorized users. * Sumarin Soonsanga sumarin.soo@biotec.or.th 1 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand