EVERNINOMICIN BIOSYNTHETIC GENES
This invention is directed to nucleic acids which encode the proteins that direct the synthesis of the orthosomycin everninomicin and to use of the nucleic acids and proteins to produce compounds exhibiting antibiotic activity based on the everninomycin structure. The DNA sequence for the gene clusters responsible for encoding everninomicin biosynthetic genes, which provide the machinery for producing everninomicin, are provided. Thus, this invention provides the nucleic acid sequences needed to synthesize novel everninomicin-related compounds based on everninomicin, arising from modifications of the DNA sequence designed to change glycosyl and modified orsellinic acid groups contained in everninomicin. A Micromonospora site-specific integrase gene is also provided, which can be incorporated in a vector for integration into any actinomycete, and, particularly into Monospora. Thus, the invention further provides methods for introducing heterologous genes into an actinomycete chromosome using this particular vector.
1 . An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 177.
2 . The polynucleotide of claim 1 which comprises the nucleotide sequence set forth in SEQ ID NO: 176.
3 . An isolated vector comprising the polynucleotide of claim 1 .
4 . The vector of claim 3 which is a plasmid.
5 . The vector of claim 3 further comprising a heterologous gene.
6 . The vector of claim 3 further comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204.
7 . The vector of claim 3 further comprising a polynucleotide comprising a nucleotide sequence selected from the group consisting of: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 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, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201 and 203.
8 . A host cell containing the vector of claim 3 .
9 . The host cell of claim 7 , which is a bacterial host cell.
10 . The host cell of claim 8 , which is an E. coli or an actinomycete.
11 . The polynucleotide of claim 1 operably associated with a transcriptional and translational control sequence.
12 . The polynucleotide of claim 1 encoding a chimeric polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 177 fused to a heterologous polypeptide.
13 . The polynucleotide of claim 12 wherein the heterologous polypeptide is a member selected from the group consisting of a poly-histidine tag, a FLAG tag, a glutathione-S-transferase (GST) tag and a myc epitope tag.
14 . The isolated polynucleotide of claim 1 consisting of the nucleotide sequence set forth in SEQ ID NO: 176.