title: Improving the N-terminal diversity of sansanmycin through mutasynthesis.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Shi
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Zhibo
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Lei
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Ningning
authors.initials: N
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Cai
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Li
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Lifei
authors.initials: L
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China.
authors.forename: Shuyi
authors.initials: S
authors.lastname: Si
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China. xieyy@imb.pumc.edu.cn.
authors.forename: Yunying
authors.initials: Y
authors.lastname: Xie
authors.affiliation: The Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing, 100050, China. binhong69@hotmail.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Hong
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D008968:Molecular Conformation; D005810:Multigene Family; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D009842:Oligopeptides; C519865:sansanmycin; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D014529:Uridine
keywords: Mutasynthesis; Novel sansanmycin analogues; m-Tyr; ssaX deletion mutant