【摘要】 螺旋霉素(SP)为16元环大环内酯类抗生素,含有螺旋霉素Ⅰ、Ⅱ和Ⅲ个组分,其结构的差异为16元内酯环的C3上分别连接羟基(SPⅠ)、乙酰基(SPⅡ)和丙酰基(SPⅢ);SPⅡ和SPⅢ是在相同的3-O-酰基转移酶催化下SPⅠ进一步酰化的产物。SPⅠ、SPⅡ和SPⅢ在生物学活性方面无大差异。为简化螺旋霉素组分,便于今后对其结构进行进一步改造,根据碳霉素和麦迪霉素生物合成中的3-O-酰基转移酶序列,设计了简并性PCR引物,并采用SON-PCR(single oligonucleotide nested PCR)方法,从螺旋霉素产生菌S.spiramyceticus F21中进行特异性扩增,获得了螺旋霉素3-O-酰基转移酶基因(sspA)及其侧翼序列,共约4.3kb(其中的3457nt DNA序列已被Genbank收录,DQ642742)。采用DNA同源双交换技术对S.spiramyceticus F21中的sspA进行了删除。对螺旋霉素原株和sspA缺失变株进行发酵产物提取和HPLC分析表明:原株中SPⅠ、SPⅡ和SPⅢ的相对含量分别为7.8%、67%和25%,变株中则分别为72%、18%和9.6%;变株主要组分为SPⅠ。螺旋霉素sspA缺失变株的获得为螺旋霉素组分简化及其衍生物的结构改造奠定了基础。更多还原

【Abstract】 Spiramycin （SP） belongs to the 16-member macrolide antibiotics. It contains three components,namely SPⅠ,SPⅡ and SPⅢ,which differ structurally in the acylation moieties on the C₃ of the lactone. The SPⅠ component contains a hydroxyl group at C₃. SPⅡ, and SPⅢ are formed by further acetylation or propionylation of the C₃ of SPⅠ,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 SPⅠ to SPⅡ and SPⅢ. 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-O-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 4.3kb 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 SPⅠ （72%）,SPⅡ （18%）, and SPⅢ（9.6%）,whereas parent strain produced SPⅠ （7.8%）,SPⅡ （67%）, and SPⅢ（25%）,respectively,demonstrating the role of sspA in the acylation of SPⅠ into SPⅡ and SPⅢ. The sspA-deleted mutant strain obtained in this study may be used for the production of SPⅠ,or may serve as a good starter for the construction of spiramycin derivatives.更多还原