Wu Lin-Zhuan, Ma Chun-Yan, Wang Yi-Guang, Dai Jian-Lu, Li Jing-Yan, Xia Huan-Zhang
Institute of Medicinal Biotechnolgy, CAMS & PUMC, Beijing 100050, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Jul;23(4):612-7.
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.
螺旋霉素（SP）属于16元大环内酯类抗生素。它含有三个组分，即SP I、SP II和SP III，它们在内酯环C3位的酰化部分结构不同。SP I组分在C3位含有一个羟基。SP II和SP III是由SP I的C3位通过相同的3 - O - 酰基转移酶（3 - O - AT）进一步乙酰化或丙酰化形成的。该研究聚焦于简化螺旋霉素组分。理论上，破坏/缺失3 - O - AT基因将减少/阻止SP I向SP II和SP III的酰化。在本研究中，分别根据嗜热栖热放线菌和弗氏链霉菌的麦迪霉素和碳霉素产生菌中3 - O - 酰基转移酶、MdmB和AcyA的保守区域设计简并引物，从螺旋链霉菌F21的螺旋霉素产生菌中扩增出一个878bp的DNA片段。对该878bp DNA片段的Blast分析表明，它编码螺旋霉素生物合成的3 - O - 酰基转移酶（3 - 0 - AT，sspA）基因。然后通过单寡核苷酸巢式PCR扩增该878bp DNA片段的侧翼区域，共获得4.3kb DNA（4.3kb片段中的3457nt已测序，并保存在GenBank DQ642742中），覆盖整个假定的3 - O - 酰基转移酶基因sspA。然后通过温度敏感质粒pKC1139介导的双交换同源重组从螺旋链霉菌F21基因组中删除sspA。通过HPLC分析比较了缺失sspA的突变菌株与亲本菌株产生的螺旋霉素组分，结果表明缺失sspA的突变菌株产生SP I（72%）、SP II（18%）和SP III（9.6%），而亲本菌株分别产生SP I（约7.8%）、SP II（67%）和SP III（25%），证明了sspA在SP I酰化为SP II和SP III中的作用。本研究获得的缺失sspA的突变菌株可用于生产SP I，或可作为构建螺旋霉素衍生物的良好起始菌株。
