title: [Deletion of spiramycin 3-O-acyltransferase gene from Streptomyces spiramyceticus F21 resulting in the production of spiramycin I as major component].
abstract: Spiramycin (SP) belongs to the 16-member macrolide antibiotics. It contains three components,namely SP I, SP II and SP III, which differ structurally in the acylation moieties on the C3 of the lactone. The SP I component contains a hydroxyl group at C3. SP II, and SP III are formed by further acetylation or propionylation of the C3 of SP I, by the same 3-O-acyltransferase (3-O-AT) . The study focused on simplifying spiramycin components. Theoretically, disruption/deletion of the 3-O-AT gene will reduce/stop the acylation of SP I to SP II and SP III. In this study, degenerated primers were designed according to the conserved regions of 3-O-acyltransferase, MdmB and AcyA in the medicamycin and carbomycin producers of S. mycarofaciens and S. thermotolerans, respectively, and an 878bp DNA fragment was amplified from the spiramycin-producer of S. spiramyceticus F21. Blast analysis of the 878bp DNA fragment suggested that it encoded the 3-O-acyltransferase (3-0-AT, sspA) gene for spiramycin biosynthesis. The flanking regions of this 878bp DNA fragment were then amplified by single-oligonucleotide-nested PCR, and a total of 4.3 kb DNA was obtained (3457nt among the 4.3kb fragment was sequenced, and deposited in GenBank DQ642742),covering the whole putative 3-O-acyltransferase gene, sspA. The sspA was then deleted from the S. spiramyceticus F21 genome by double cross-over homologous recombination, mediated by temperature-sensitive plasmid pKC1139. A comparison was done of the components of spiramycins produced by the sspA-deleted mutant strain with that of the parent strain by HPLC analysis, which showed that sspA-deleted mutant produced SP I (72%), SP II (18%), and SP III (9.6%), whereas parent strain produced SP I (7.8%), SP II (67%), and SP III (25%), respectively, demonstrating the role of ssp A in the acylation of SP I into SP II and SP III. The ssp A-deleted mutant strain obtained in this study may be used for the production of SP I, or may serve as a good starter for the construction of spiramycin derivatives.
authors.affiliation: Institute of Medicinal Biotechnolgy, CAMS & PUMC, Beijing 100050, China.
authors.forename: Lin-Zhuan
authors.initials: LZ
authors.lastname: Wu
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Ma
authors.forename: Yi-Guang
authors.initials: YG
authors.lastname: Wang
authors.forename: Jian-Lu
authors.initials: JL
authors.lastname: Dai
authors.forename: Jing-Yan
authors.initials: JY
authors.lastname: Li
authors.forename: Huan-Zhang
authors.initials: HZ
authors.lastname: Xia
mesh_terms: D000217:Acyltransferases / Q000235:genetics*; D000617:Aminoglycosides / Q000096:biosynthesis*; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
chemical_list: D000617:Aminoglycosides; C017186:demycarosylturimycin H; D000217:Acyltransferases