Functional Analysis of Genes Encoding Glycosyltransferases Involved in the Biosynthesis of Deoxysugar-containing Antibiotics

菌株Tu2717产生的URDs是一类安环素类抗生素。主要化合物乌达霉素A （urdamycin A, URD-A）在C-9位点上以三糖单位（D-olivose-L-rhodinose-D-olivose）连接C′糖苷，在C-12b位点上以单L-rhodinose为O~糖苷。S. fradiae Tu2717的一个30 kb的基因组区域携带URD生物合成基因簇（URD簇），包括脱氧糖形成酶UrdG、UrdH、UrdS、UrdT、UrdQ、UrdZ1和UrdZ3的基因。他们推断的功能使我们能够将基因功能分配到d-葡萄糖-1-磷酸衍生的d-葡萄糖和l -罗丹糖的每个生物合成步骤，分支在ndp -4-酮葡萄糖-2,6-二脱氧-d -葡萄糖的阶段，该阶段由Urd-R转化为ndp -d -葡萄糖或由UrdQ， UrdZl和UrdZ3转化为ndp - l -罗丹糖。urd集群提供了四个糖基转移酶（GT）基因，urdGT1a、urdGT1b、urdGT1c和urdGT2，每个基因都被分配到urd - a生物合成中的特定GT反应中。特别有趣的是urdGT1b和urdGT2，尽管它们的D-rhodinose （UrdGT1c）和D-olivose （urdGT1b）的转移酶功能不同，但它们在氨基酸水平上惊人地相似（91%的同源性）。它们还具有区域特异性和机制特异性，即ci-(1,3)-糖基化UrdGTlc β-(1,4)-糖基化UrdGTlb。构建了一系列嵌合的GT基因，并在缺乏GT1b/GTc活性的S. fradiae突变体中表达，以寻找决定底物特异性的区域。n端附近有一个31个氨基酸的区域，与18个氨基酸不同，控制糖和受体的特异性。该区域的代谢工程，包括DNA重组，产生了新的GT特异性，导致非自然的URD衍生物，包括URD- p具有支链三核苷酸链。

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.