|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
URDs produced by S. fradiae Tu2717 are a class of angucycline antibiotics. The principal compound, urdamycin A (URD-A), bears trisaccharide unit (D-olivose-L-rhodinose-D-olivose) attached C'glycosidically to the polyketide aglycone at C-9, and single L-rhodinose as O~glycoside at C-12b. A 30-kb genomic region of S. fradiae Tu2717 carries the URD biosynthetic gene cluser (the urd cluster), including the genes for the deoxysugar-forming enzymes, UrdG, UrdH, UrdS, UrdT, UrdQ, UrdZ1, and UrdZ3. Their deduced functions allowed us to assign the gene functions to each of the biosynthetic steps at D-olivose and L-rhodinose derived from D-glucose-1-phosphate, branching at the stage of NDP-4-ketoglucose-2,6-dideoxy-D-glucose which is converted either to NDP-D-olivose by Urd-R or to NDP-L-rhodinose by UrdQ, UrdZl, and UrdZ3. The urd cluster provides four glycosyltransferase (GT) genes, urdGT1a, urdGT1b, urdGT1c, and urdGT2, each of which was assigned to a specific GT reaction in URD-A biosynthesis. Particularly interesting are urdGT1b and urdGT2, since they are strikingly similar (91 % identity at the amino acidlevel) despite of their distinct transferase functions of D-rhodinose (UrdGT1c) and D-olivose (UrdGT1b). They also possess regio-and mechanistic specificities ci-(1,3)-glycosylation for UrdGTlc β-(1,4)-glycosylation for UrdGTlb. A series of their chimeric GT genes were constructed, and expressed in S. fradiae mutants lacking GT1b/GTc activity to find the region determining the substrate specificities. A 31-amino acid region close to the N-terminus, which differs 18 amino acids, was identified to control both sugar and acceptor specificities. Metabolic engineering to the region, including DNA shuffling, gave rise to the novel GT specificities leading to unnatural URD derivatives, including URD-P possessing a branched trisaccahradide chain.