Polypeptides Having Antimicrobial Activity and Polynucleotides Encoding Same

Abstract

The present invention relates to isolated polypeptides having antimicrobial activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

1 . A polypeptide having antimicrobial activity, which comprises an amino acid sequence which has at least 80% identity with amino acids 1 to 40 of SEQ ID NO: 2: 
       
         
           
                 
               
                   G-X 1 -G- C -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 - C -H-X 12 - 
                 
                     
                 
                   X 13 - C -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -G-G-X 21 - C -X 22 - 
                 
                     
                 
                   X 23 -X 24 -X 25 -X 26 -X 27 - C -X 28 - C -X 29 ; 
                 
             
                
                
                
                
                
               
            
           
         
       
       wherein
 X 1 =F, L, W or I; preferably X 1 =F; 
 X 2 =N, R, Q, V, G, S, A, K, L, M, D, H or Y; preferably X 2 =N, R, Q, V, G, S, A, K or Y; 
 X 3 =G, R, A or K; preferably X 3 =G; 
 X 4 =P, A, L, V, K or R; preferably X 4 =P, K or R; 
 X 5 =W or R; 
 X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or Y; preferably X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, V or Y; 
 X 7 =E, G, A, L, C, Q or S; preferably X 7 =E, G or S; 
 X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M, A, C or R; preferably X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or R; 
 X 9 =D or P; preferably X 9 =D; 
 X 10 =M, R, S, V, A, F, G, L, T, Y, W, E or K; preferably X 10 =M, R, S, V, G, Y, L, F, T, W or K; 
 X 11 =Q, R, L, F, G, H, S, A, C, I, K, M, P, T, V, W or Y; preferably X 11 =Q, R, L, F, G, H, S, K or Y; 
 X 12 =N, R, I, Y, V, K, T, Q, S, A, W, E or H; 
 X 13 =H, A, F, Q, T, V or L; preferably X 13 =H or L; 
 X 14 =K, Q or R; preferably X 14 =K or R; 
 X 15 =S, A, V, N or F; 
 X 16 =I, L, M, T, W or V; preferably X 16 =I, L or V; 
 X 17 =K, T or R; 
 X 18 =G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably X 18 =G, H, R, K or N; 
 X 19 =Y, H, K, L, M, N, Q, S, V or R; preferably X 19 =Y or R; 
 X 20 =K, F, H, T, C or R; preferably X 20 =K or R; 
 X 21 =Y, F, R, A, H, L, M, S or W; preferably X 21 =Y, F, R or W; 
 X 22 =A, K, N, Q, T, E, H, I, R, S, V, G or Y; preferably X 22 =A, K, N, Q, T, S or Y; 
 X 23 =K, R or T; preferably X 23 =K or R; 
 X 24 =G, K, Q, E, N, S, T, A or R; preferably X 24 =G, K, Q, A or R; 
 X 25 =G, K, H, W or R; preferably X 25 =G, K or R; 
 X 26 =F, A, H, I, M, V, W, R or L; preferably X 26 =F or L; 
 X 27 =V, L, M, I, K, Q, R or T; preferably X 27 =V, L, M or T; 
 X 28 =K, H, N or R; preferably X 28 =K or R; 
 X 29 =Y, I, YRCG or YR; preferably X 29 =Y or YR; 
 and which has less than 100% identity with amino acids 1 to 40 of SEQ ID NO: 1. 
 
     
     
         2 . The polypeptide of  claim 1 , wherein the amino acid sequence has at least 85% identity with amino acids 1 to 40 of SEQ ID NO: 2, preferably at least 90% identity, or at least 95% identity with amino acids 1 to 40 of SEQ ID NO: 2. 
     
     
         3 . The polypeptide of  claim 1 , which comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         4 . The polypeptide of  claim 1 , which comprises the amino acid sequence of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. 
     
     
         5 . The polypeptide of  claim 1 , which consists of the amino acid sequence of SEQ ID NO: 2; or a fragment thereof having antimicrobial activity and comprising 6 cysteine residues. 
     
     
         6 . The polypeptide of  claim 5 , which consists of the amino acid sequence of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274; or a fragment thereof having antimicrobial activity and comprising 6 cysteine residues. 
     
     
         7 . A composition comprising a polypeptide of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         8 . The composition of  claim 7 , which further comprises an additional biocidal agent. 
     
     
         9 . A detergent composition comprising a surfactant and the polypeptide of  claim 1 . 
     
     
         10 . An animal feed additive comprising
 (a) a polypeptide of  claim 1 ; and   (b) a fat soluble vitamin, and/or   (c) a water soluble vitamin, and/or   (d) a trace mineral, and/or   (e) a macro mineral.   
     
     
         11 . The animal feed additive of  claim 10 , which further comprises phytase, xylanase, galactanase, and/or beta-glucanase. 
     
     
         12 . An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising a polypeptide of  claim 1 . 
     
     
         13 . A method for killing or inhibiting growth of microbial cells comprising contacting the microbial cells with a polypeptide of  claim 1 . 
     
     
         14 . A method of treating a microbial infection, comprising administering to a human or animal a polypeptide of  claim 1  in an amount effective to treat the microbial infection. 
     
     
         15 . A method of treating tuberculosis, comprising administering to a human or animal a polypeptide of  claim 1  in an amount effective to treat the tuberculosis. 
     
     
         16 . A polynucleotide comprising a nucleotide sequence which encodes a polypeptide of any of  claim 1 . 
     
     
         17 . A nucleic acid construct comprising the polynucleotide of  claim 16  operably linked to one or more control sequences that direct the production of the polypeptide in an expression host. 
     
     
         18 . A recombinant expression vector comprising the nucleic acid construct of  claim 17 . 
     
     
         19 . A recombinant host cell comprising the nucleic acid construct of  claim 17 . 
     
     
         20 . A method for producing a polypeptide having antimicrobial activity, comprising
 (a) cultivating the recombinant host cell of  claim 19  under conditions conducive for production of the polypeptide; and   (b) recovering the polypeptide.