Improving the N-terminal diversity of sansanmycin through mutasynthesis.
Improving the N-terminal diversity of sansanmycin through mutasynthesis.
复制标题
通过诱变合成提高山散霉素的 N 末端多样性。
DOI：
10.1186/s12934-016-0471-1
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发表时间：
2016-05-06
影响因子：
6.4
通讯作者：
Hong B
中科院分区：
文献类型：
--
作者：
Shi Y;Jiang Z;Lei X;Zhang N;Cai Q;Li Q;Wang L;Si S;Xie Y;Hong B
三联霉素是尿苷肽抗生素（UPAs），它是转位酶I （MraY）的抑制剂，阻断细菌细胞壁的生物合成。对铜绿假单胞菌和结核分枝杆菌具有良好的抑菌活性。根据非核糖体肽合成酶（NRPSs）催化的pacidamycin的生物合成途径，对三霉素的生物合成基因簇进行了表征，并阐明了其主要的生物合成途径。Sananmycin A是Streptomyces sp. SS（野生型菌株）的主要化合物，它在四肽链的n端含有一个非蛋白氨基酸-间酪氨酸（m-Tyr）。采用λ-RED介导的PCR靶向法构建ssaX缺失突变体SS/XKO，并进行PCR和southern blot验证。ssaX的破坏完全消除了sansanmycin A的产生，体内和体外的补体均能恢复sansanmycin A的产生，并且ssaX的过表达明显使sansanmycin A的产生增加了20%。从ssaX缺失突变体发酵培养中鉴定出6个新化合物。通过突变合成获得了一些新的三霉素类似物，并通过电喷雾电离质谱（ESI-MS）和核磁共振（NMR）对共有10个三霉素类似物MX-1 ~ MX-10进行了纯化和鉴定。对这些三霉素类似物的生物测定表明，三霉素MX-1、MX-2、MX-4、MX-6和MX-7对结核分枝杆菌H37Rv以及多重耐药（MDR）和广泛耐药（XDR）菌株的效力与三霉素A相当。此外，三霉素MX-2和MX-4表现出比三霉素a更好的稳定性。我们证明了SsaX通过基因缺失和互补在体内负责m-Tyr的生物合成。通过对ssaX缺失突变体SS/XKO的诱变获得了20个新的三霉素类似物，并对其中10个进行了纯化和结构鉴定。其中，MX-2和MX-4表现出较好的抗mdr和抗xdr结核活性，且稳定性优于三霉素a。这些结果表明，ssaX缺失突变体SS/XKO是扩展UPAs n端多样性的合适宿主，有可能产生更多具有更高活性和/或其他特性的新化合物。本文的在线版本（doi:10.1186/s12934-016-0471-1）包含补充材料，可供授权用户使用。
Sansanmycins are uridyl peptide antibiotics (UPAs), which are inhibitors of translocase I (MraY) and block the bacterial cell wall biosynthesis. They have good antibacterial activity against Pseudomonas aeruginosa and Mycobacterium tuberculosis strains. The biosynthetic gene cluster of sansanmycins has been characterized and the main biosynthetic pathway elucidated according to that of pacidamycins which were catalyzed by nonribosomal peptide synthetases (NRPSs). Sananmycin A is the major compound of Streptomyces sp. SS (wild type strain) and it bears a non-proteinogenic amino acid, meta-tyrosine (m-Tyr), at the N-terminus of tetrapeptide chain. ssaX deletion mutant SS/XKO was constructed by the λ-RED mediated PCR targeting method and confirmed by PCR and southern blot. The disruption of ssaX completely abolished the production of sansanmycin A. Complementation in vivo and in vitro could both recover the production of sansanmycin A, and the overexpression of SsaX apparently increased the production of sansanmycin A by 20 %. Six new compounds were identified in the fermentation culture of ssaX deletion mutant. Some more novel sansanmycin analogues were obtained by mutasynthesis, and totally ten sansanmycin analogues, MX-1 to MX-10, were purified and identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The bioassay of these sansanmycin analogues showed that sansanmycin MX-1, MX-2, MX-4, MX-6 and MX-7 exhibited comparable potency to sansanmycin A against M. tuberculosis H37Rv, as well as multi-drug-resistant (MDR) and extensive-drug-resistant (XDR) strains. Moreover, sansanmycin MX-2 and MX-4 displayed much better stability than sansanmycin A. We demonstrated that SsaX is responsible for the biosynthesis of m-Tyr in vivo by gene deletion and complementation. About twenty novel sansanmycin analogues were obtained by mutasynthesis in ssaX deletion mutant SS/XKO and ten of them were purified and structurally identified. Among them, MX-2 and MX-4 showed promising anti-MDR and anti-XDR tuberculosis activity and greater stability than sansanmycin A. These results indicated that ssaX deletion mutant SS/XKO was a suitable host to expand the diversity of the N-terminus of UPAs, with potential to yield more novel compounds with improved activity and/or other properties. The online version of this article (doi:10.1186/s12934-016-0471-1) contains supplementary material, which is available to authorized users.