Method for isolation of biosynthesis genes for bioactive molecules

Abstract

Degenerate primers which hybridize with various classes of antibiotic biosynthesis genes were used to amplify fragments of DNA from soil and lichen extracts. Cloning and sequencing of the amplified products showed that these products included a variety of novel and previously uncharacterized antibiotic biosynthesis gene sequences, the products of which have the potential to be active as antibiotics, immunosuppressors, antitumor agents, etc. Thus, antibiotic biosynthesis genes can be recovered from soil or lichens by a combining a sample with a pair of amplification primers under conditions suitable for polymerase chain reaction amplification, wherein the primer set is a degenerate primer set selected to hybridize with conserved regions of known antibiotic biosynthetic pathway genes, for example Type I and Type II polyketide synthase genes, isopenicillin N synthase genes, and peptide synthetase genes; cycling the combined sample through a plurality of amplification cycles to amplify DNA complementary to the primer set; and isolating the amplified DNA.

We claim:  
     
         1 . A method for recovery of antibiotic biosynthetic DNA from humic materials or lichen comprising the steps of: 
 (a) combining a humic or lichen-derived sample with a set of amplification primers under conditions suitable for polymerase chain reaction amplification, wherein the primer set is a degenerate primer set selected to hybridize with conserved regions of antibiotic biosynthetic gene;    (b) cycling the combined sample through a plurality of amplification cycles to amplify DNA complementary to the primer set; and    (c) isolating the amplified DNA.    
     
     
         2 . The method according to  claim 1 , wherein the primer set hybridizes with a polyketide synthase gene.  
     
     
         3 . The method according to  claim 2 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 1 and 2.  
     
     
         4 . The method according to  claim 2 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 3 and 4.  
     
     
         5 . The method according to  claim 2 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 5 and 6.  
     
     
         6 . The method according to  claim 2 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 11 and 12.  
     
     
         7 . The method according to  claim 1 , wherein the primer set hybridizes with a isopenicillin N synthase gene.  
     
     
         8 . The method according to  claim 7 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 7 and 8.  
     
     
         9 . The method according to  claim 1 , wherein the primer set hybridizes with a peptide synthetase gene.  
     
     
         10 . The method according to  claim 9 , wherein the primer set comprises primers having the sequence set forth in SEQ ID Nos. 9 and 10.  
     
     
         11 . The method according to  claim 1 , wherein the sample comprises DNA extracted from a soil sample.  
     
     
         12 . The method according to  claim 1 , wherein the sample is a lichen-derived sample.  
     
     
         13 . The method according to  claim 1 , further comprising the steps of cloning the isolated DNA into a host organism, and isolating the cloned DNA.  
     
     
         14 . The method according to  claim 13 , wherein the host organism is  E. coli.    
     
     
         15 . An oligonucleotide primer having the sequence as defined in any of Seq. ID. Nos. 1 through 8.  
     
     
         16 . A composition comprising two oligonucleotide primers having the sequence as defined in Seq. ID Nos. 1 and 2; 3 and 4; 5 and 6; or 7 and 8.  
     
     
         17 . A polynucleotide comprising a region having the sequence given by any of sequence ID Nos. 13, 15, 17, 19, 21, 23, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 or 93.  
     
     
         18 . A biosynthetic polypeptide encoded by a polynucleotide comprising a region having the sequence given by any of sequence ID Nos. 13, 15, 17, 19, 21, 23, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 81, 83, 85, 87, 89, 91 or 93.  
     
     
         19 . The biosynthetic polypeptide of  claim 18 , wherein the polypeptide has the amino acid sequence given by any of Sequence ID Nos. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 3,4 3,6 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 or 94.