Mitomycin biosynthetic gene cluster

Abstract

The invention provides a biosynthetic gene cluster for mitomycin, as well as methods of using gene(s) within the cluster to alter biosynthesis.

What is claimed is:  
     
         1 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a mitomycin biosynthetic gene cluster, a variant or a fragment thereof.  
     
     
         2 . The isolated and purified nucleic acid molecule of  claim 1  which encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.  
     
     
         3 . The isolated and purified nucleic acid molecule of  claim 1  which encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.  
     
     
         4 . The isolated and purified nucleic acid molecule of  claim 1  which is from Streptomyces spp.  
     
     
         5 . An expression cassette comprising the nucleic acid molecule of  claim 1 ,  2  or  3  operably linked to a promoter functional in a host cell.  
     
     
         6 . A recombinant bacterial host cell in which at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster is disrupted to as to result in a recombinant host cell that produces altered levels of mitomycin relative to a corresponding nonrecombinant bacterial host cell.  
     
     
         7 . The recombinant host cell of  claim 6  in which mitomycin levels are increased.  
     
     
         8 . The recombinant host cell of  claim 6  in which mitomycin levels are decreased.  
     
     
         9 . The host cell of  claim 6  wherein the nucleic acid molecule which is disrupted encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.  
     
     
         10 . The host cell of  claim 6  wherein the nucleic acid molecule which is disrupted encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.  
     
     
         11 . A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster operably linked to a promoter functional in the host cell.  
     
     
         12 . The recombinant host cell of  claim 11  in which mitomycin levels are increased.  
     
     
         13 . The recombinant host cell of  claim 11  in which mitomycin levels are decreased.  
     
     
         14 . The host cell of  claim 11  wherein the genome is augmented with a nucleic acid molecule that encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.  
     
     
         15 . The host cell of  claim 11  wherein the genome is augmented with a nucleic acid molecule that encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.  
     
     
         16 . A recombinant host cell comprising a mitomycin biosynthetic gene cluster, the genome of which is augmented by a recombinant nucleic acid molecule, wherein the recombinant nucleic acid does not comprise a mitomycin biosynthetic gene, and wherein the recombinant host cell produces a biologically active agent that is not produced by the corresponding non-recombinant host cell.  
     
     
         17 . A product produced by the recombinant host cell of  claim 6  or  11  which is not produced by the corresponding non-recombinant host cell.  
     
     
         18 . The product of  claim 17  which comprises a biologically active agent.  
     
     
         19 . The product of  claim 18  which is a mitomycin.  
     
     
         20 . The product of  claim 18  is not a mitomycin.  
     
     
         21 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes polyketide biosynthetic enzymes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.  
     
     
         22 . An isolated and purified polypeptide comprising MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.  
     
     
         23 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising sugar metabolism genes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.  
     
     
         24 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes an aminoDAHP synthase from Streptomyces strains that produce mitomycin.  
     
     
         25 . A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes is disrupted so as to result in a recombinant host cell that produces altered polyketide levels or polyketides of altered composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74  
     
     
         26 . A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes is disrupted so as to result in a recombinant host cell that produces altered sugar levels or molecules with altered sugar composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.  
     
     
         27 . A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.  
     
     
         28 . A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.  
     
     
         29 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence that hybridizes under hybridizing conditions to a nucleic acid segment comprising SEQ ID NO:96, or a fragment thereof.  
     
     
         30 . The isolated and purified nucleic acid molecule of  claim 29  which is plant nucleic acid.  
     
     
         31 . The isolated and purified nucleic acid molecule of  claim 29  which is prokaryotic nucleic acid.  
     
     
         32 . A method to introduce exogenous DNA into a refractory Streptoinyces strain, comprising: 
 a) contacting a bacterial donor cell comprising a conjugative plasmid with a Streptomyces cell so as to yield a transformed Streptomyces cell comprising at least a portion of the plasmid; and    b) identifying the transformed Streptomyces cell.    
     
     
         33 . The method of  claim 32  wherein the Streptomyces strain produces a mitomycin.  
     
     
         34 . A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster, comprising: 
 a) contacting a sample comprising nucleic acid with an amount of a probe comprising at least a portion of a nucleic acid molecule comprising a mitomycin gene so as to form a complex;    b) detecting the presence or absence of the complex.    
     
     
         35 . A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster comprising: 
 a) contacting a sample comprising nucleic acid with at least one oligonucleotide under conditions effective to amplify the nucleic acid so as to yield an amplification product, wherein the oligonucleotide specifically hybridizes to nucleic acid comprising a mitomycin gene cluster; and    b) detecting or determining the presence or absence of the product.    
     
     
         36 . The method of  claim 34  or  35  wherein the sample is obtained from a plant.  
     
     
         37 . The method of  claim 34  or  35  wherein the sample is obtained from a microorganism.  
     
     
         38 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a gene product selected from MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, MmcW, MmcX, MmcY, or any combination thereof.  
     
     
         39 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene necessary for mitomycin biosynthesis.  
     
     
         40 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene for mitomycin transport.  
     
     
         41 . An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide that regulates mitomycin biosynthesis or resistance.  
     
     
         42 . A method for preparing a compound or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of  claim 6 ,  11  or  16  in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the compound relative to the level of the compound produced by the corresponding non-recombinant host cell.  
     
     
         43 . A method for preparing a mitomycin or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of  claim 6 ,  11  or  16  in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the mitomycin relative to the level of the mitomycin produced by the corresponding non-recombinant host cell.  
     
     
         44 . A product produced by the recombinant host cell of  claim 16  which is a mitomycin.  
     
     
         45 . A product produced by the recombinant host cell of  claim 16  which is not a mitomycin.  
     
     
         46 . An isolated and purified nucleic acid molecule comprising at least a fragment of a nucleic acid sequence comprising a mitomycin biosynthetic gene cluster (mit/mmc), which fragment encodes an enzyme that during the biosyntheis of mitomycin modifies mitosane, and which fragment has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, 68, or comprising the complement of the fragment.  
     
     
         47 . The isolated and purified nucleic acid molecule of  claim 46  which encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO: 104, or MitF having SEQ ID NO: 102.  
     
     
         48 . The isolated and purified nucleic acid molecule of  claim 46  which encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO: 124, MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN having SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.  
     
     
         49 . The isolated and purified nucleic acid molecule of  claim 46  which is from a naturally-occurring Streptomyces spp.  
     
     
         50 . An expression cassette comprising the nucleic acid molecule of  claim 46  operably linked to a promoter functional in a host cell.  
     
     
         51 . A recombinant host cell comprising a recombinant nucleic acid molecule comprising at least a fragment of a mitomycin biosynthetic gene cluster (mit/mmc) operably linked to a promoter functional in the host cell, which fragment encodes an enzyme that during the biosynthesis of mitomycin modifies mitosane, and which fragment has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, 68, or comprising the complement of the fragment.  
     
     
         52 . The recombinant host cell of  claim 51  in which the levels of the enzyme are increased.  
     
     
         53 . The recombinant host cell of  claim 51  in which the levels of the enzyme are decreased.  
     
     
         54 . The recombinant host cell of  claim 51  wherein the fragment encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO: 104, or MitF having SEQ ID NO: 102.  
     
     
         55 . The recombinant host cell of  claim 51  wherein the fragment encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO: 124, MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN having SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.  
     
     
         56 . The isolated and purified nucleic acid molecule of  claim 46  wherein the enzyme is a hydroxylase, a reductase, a dehydrogenase, methyltransferase, or converts a carboxyl group to a methyl group.  
     
     
         57 . The isolated and purified nucleic acid molecule of  claim 46  wherein the fragment comprises SEQ ID NO:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62 or 68.  
     
     
         58 . A method to prepare an enzyme that catalyzes a step in mitomycin biosynthesis: expressing a recombinant DNA molecule in a host cell so as to yield an enzyme that catalyzes a step in mitomycin biosynthesis, wherein the recombinant DNA molecule comprises a promoter operably linked to a DNA sequence which encodes an enzyme that modifies mitosane, and wherein the DNA sequence has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, or 68.