Improving the N-terminal diversity of sansanmycin through mutasynthesis

Yuanyuan Shi, Zhibo Jiang, Xuan Lei, Ningning Zhang, Qiang Cai, Qinglian Li, Lifei Wang, Shuyi Si, Yunying Xie, Bin Hong

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.