Mutant polypeptides and uses thereof

Abstract

The present disclosure provides novel polypeptides with 3-buten-2-ol dehydratase activity, polypeptides with catalytic activity in the conversion of 3-methyl-3-buten-2-ol to isoprene, and crystal structure data for one of such polypeptides. Methods of making and using the polypeptides and their related crystal structure data are also provided.

1 .- 20 . (canceled) 
     
     
         21 . A composition comprising 1,3-butadiene and a polypeptide comprising an amino acid sequence with at least 90% amino acid sequence homology to SEQ ID NO: 1, wherein said amino acid sequence comprises one to five mutations at the following X positions of SEQ ID NO: 1: 
       
         
           
                 
               
                   R 1-95 X 96 R 97-98 X 99 R 100-122 X 123 R 124-186 X 187 R 188-203   
                 
                     
                 
                   X 204 R 205-211 X 212 R 213-272 X 273 X 274 X 275 R 276-323   
                 
                     
                 
                   X 324 R 325-327 X 328 R 329-359 X 360 R 361-365 X 366 R 367-381   
                 
                     
                 
                   X 382 R 383-398 , 
                 
             
                
                
                
                
                
                
                
               
            
           
         
         wherein: 
         X96 is mutated to a different amino acid selected from L, I, M, A, G, and V; 
         X99 is mutated to a different amino acid selected from L, I, M, A, G, and V; 
         X123 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X187 is mutated to a different amino acid selected from L, M, A, G, and V; 
         X204 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X212 is mutated to a different amino acid selected from F, Y, and W; 
         X273 is mutated to a different amino acid selected from C, M, S, and T; 
         X274 is mutated to a different amino acid selected from F, Y, and W; 
         X275 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X324 is mutated to a different amino acid selected from L, I, M, G, V, E, D, N, and Q; 
         X328 is mutated to a different amino acid selected from I, M, A, G, and V; 
         X360 is mutated to a different amino acid selected from F, Y, and W; 
         X366 is mutated to a different amino acid selected from L, I, M, A, G, V, C, and T; 
         X382 is mutated to a different amino acid selected from Y and W; and each R is the same as the corresponding amino acid in SEQ ID NO: 1, with or without an N-terminal signal peptide, and with or without an N-terminal methionine, 
         wherein the polypeptide has an activity in converting 3-buten-2-ol to 1,3-butadiene and/or in converting 3-methyl-3-buten-2-ol to isoprene, 
         and provided that the mutations chosen from one or more of positions X96, X99, X123, and X187 are not the only mutations of SEQ ID NO: 1. 
       
     
     
         22 . The composition of claim  1 , wherein the amino acid sequence of the polypeptide has at least 95% amino acid sequence homology to SEQ ID NO: 1. 
     
     
         23 . The composition of claim  1 , wherein the amino acid sequence of the polypeptide comprises one to three mutations at the following X positions of SEQ ID NO: 1: 
       
         
           
                 
               
                   R 1-95 X 96 R 97-98 X 99 R 100-122 X 123 R 124-186 X 187 R 188-203   
                 
                     
                 
                   X 204 R 205-211 X 212 R 213-272 X 273 X 274 X 275 R 276-323 X 324   
                 
                     
                 
                   R 325-327 X 328 R 329-359 X 360 R 361-365 X 366 R 367-381 X 382   
                 
                     
                 
                   R 383-398   
                 
             
                
                
                
                
                
                
                
               
            
           
         
         wherein: 
         X96 is mutated to a different amino acid selected from L, I, M, A, G, and V; 
         X99 is mutated to a different amino acid selected from L, I, M, A, G, and V; 
         X123 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X187 is mutated to a different amino acid selected from L, M, A, G, and V; 
         X204 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X212 is mutated to a different amino acid selected from F, Y, and W; 
         X273 is mutated to a different amino acid selected from C, M, S, and T; 
         X274 is mutated to a different amino acid selected from F, Y, and W; 
         X275 is mutated to a different amino acid selected from L, I, M, A, and G; 
         X324 is mutated to a different amino acid selected from L, I, M, G, V, E, D, N, and Q; 
         X328 is mutated to a different amino acid selected from I, M, A, G, and V; 
         X360 is mutated to a different amino acid selected from F, Y, and W; 
         X366 is mutated to a different amino acid selected from L, I, M, A, G, V, C, and T; 
         X382 is mutated to a different amino acid selected from Y and W; and each R is the same as the corresponding amino acid in SEQ ID NO: 1, with or without an N-terminal signal peptide, and with or without an N-terminal methionine, 
         wherein the polypeptide is capable of converting 3-buten-2-ol to 1,3-butadiene and/or converting 3-methyl-3-buten-2-ol to isoprene, 
         and provided that the mutations chosen from one or more of positions X96, X99, X123, and X187 are not the only mutations in SEQ ID NO: 1. 
       
     
     
         24 . The composition of claim  1 , wherein the polypeptide is mutated at X187 to a different amino acid selected from L, M, A, G, and V. 
     
     
         25 . The composition of claim  1 , wherein the polypeptide is mutated at X328 to a different amino acid selected from I, M, A, G, and V. 
     
     
         26 . The composition of claim  1 , wherein the polypeptide is mutated at X382 to a different amino acid selected from Y and W. 
     
     
         27 . The composition of claim  1 , wherein the polypeptide comprises the following two mutations: L328V and F382W. 
     
     
         28 . The composition of claim  1 , wherein the polypeptide comprises the following three mutations: I187M, L328V, and F382W. 
     
     
         29 . The composition of claim  1 , wherein the polypeptide has an activity in the catalysis of the dehydration of 3-buten-2-ol to 1,3-butadiene that is at least 80% of that of a polypeptide consisting of SEQ ID NO: 1, 4, 5, 7, or 8, wherein said activity is observed in at least one activity assay.