Evolution of bt toxins

Abstract

The disclosure provides amino acid sequence variants of Bacillus thuringiensis (Bt) toxins and methods of producing the same. Some aspects of this disclosure provide methods for generating Bt toxin variants by continuous directed evolution. Some aspects of this disclosure provide compositions and methods for pest control using the disclosed variant Bt toxins.

1 - 59 . (canceled) 
     
     
         60 . A method for producing a modified pesticidal protein, the method comprising introducing one or more mutations into a nucleic acid sequence encoding a receptor-binding domain of a pesticidal protein by phage-assisted continuous evolution (PACE) to obtain a modified nucleic acid sequence encoding the modified pesticidal protein, wherein the modified pesticidal protein binds a non-cognate receptor of a target pest. 
     
     
         61 . The method of  claim 60 , wherein the pesticidal protein is a  Bacillus thuringiensis  (Bt) toxin. 
     
     
         62 . The method of  claim 60 , wherein the non-cognate receptor comprises a toxin binding region (TBR). 
     
     
         63 . The method of  claim 60 , wherein the modified pesticidal protein comprises an amino acid sequence that is at least 90% amino acid sequence identical to a Cry 1 Ac protein having the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         64 . The method of  claim 60 , wherein the one or more mutations in the nucleic acid sequence result in a nonsense mutation, missense mutation, synonymous mutation, or non-synonymous amino acid substitution. 
     
     
         65 . The method of  claim 60 , wherein the modified pesticidal protein kills the target pest. 
     
     
         66 . The method of  claim 60 , wherein the pesticidal protein is Cry1Ac (SEQ ID NO: 1). 
     
     
         67 . The method of  claim 66 , wherein the modified pesticidal protein binds the non-cognate receptor of the target pest with higher affinity than Cry 1 Ac. 
     
     
         68 . The method of  claim 66 , wherein Cry1Ac does not bind to the non-cognate receptor of the target pest. 
     
     
         69 . The method of  claim 60 , wherein the receptor binding domain comprises an amino acid sequence that is at least 90% identical to amino acid residues 275-462 of SEQ ID NO: 1. 
     
     
         70 . The method of  claim 60 , wherein the non-cognate receptor is a cadherin or a cadherin-like receptor. 
     
     
         71 . The method of  claim 70 , wherein the non-cognate receptor is a Trichoplusia ni cadherin receptor or a  Chrysodeixis includens  cadherin receptor. 
     
     
         72 . The method of  claim 60 , wherein the modified pesticidal protein binds to a receptor comprising an amino acid sequence represented by SEQ ID NO: 2. 
     
     
         73 . The method of  claim 60 , wherein the modified pesticidal protein binds to a receptor comprising an amino acid sequence represented by SEQ ID NO: 5. 
     
     
         74 . The method of  claim 60 , wherein the modified pesticidal protein binds to a receptor comprising an amino acid sequence represented by SEQ ID NO: 6. 
     
     
         75 . The method of  claim 60 , wherein the pest is a caterpillar, moth, beetle, fly, gnat, or mosquito. 
     
     
         76 . The method of  claim 60 , wherein the pest is from insect order  Lepidoptera , Coleoptera, Diptera, or Hemiptera. 
     
     
         77 . The method of  claim 60 , wherein the modified pesticidal protein retains ability to bind to its native receptor of the target pest. 
     
     
         78 . The method of  claim 77 , wherein the native receptor is ABC transporter ABCC2 or aminopeptidase N (APN1). 
     
     
         79 . The method of  claim 60 , wherein the method further comprises reverting one or more mutations of the modified pesticidal protein obtained using PACE.