Improving the N-terminal diversity of sansanmycin through mutasynthesis

Abstract

BackgroundSansanmycins 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.ResultsssaX 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0471-1) contains supplementary material, which is available to authorized users.

New tuberculosis drug leads from naturally occurring compounds

“…98,100 Simple semi-synthetic and biosynthetic modifications to the sansanmycin natural product has resulted in derivatives with greater activity against virulent M. tuberculosis, including MDR and XDR strains. 101…”

Synthesis of functionalized 2,3-diaminopropionates and their potential for directed monobactam biosynthesis

“…53,54 In NRPS biosynthesis, uorinated amino acids have been successfully incorporated into peptidyl natural products, however these examples consist almost exclusively of halogen-substituted aromatic a-amino acids. 25,55,56 Owing to the much simpler installation of C-sp 2 -F bonds, these biosynthetic precursors are either commercially available or prepared in few synthetic steps. For instance, in order to isolate a uorinated analog of balhimycin, Süssmuth et al applied a three-step synthetic sequence to access racemic uorinated beta-hydroxy tyrosine, which was required in multigram quantities to isolate the mutasynthetic product.…”

“…20–22 Contemporary approaches in the field of NRPS-based mutasynthesis, however, are usually limited to substituted aromatic non-proteinogenic building blocks such as hydroxyphenylglycines, β-hydroxytyrosine or salicylates, where synthetic analogs are readily available. 20,23–26…”

“…[20][21][22] Contemporary approaches in the eld of NRPSbased mutasynthesis, however, are usually limited to substituted aromatic non-proteinogenic building blocks such as hydroxyphenylglycines, b-hydroxytyrosine or salicylates, where synthetic analogs are readily available. 20,[23][24][25][26] In this study we aimed to extend this concept through supplementation of non-native derivatives of L-Dap to P. acidophila DsulG. Here, these synthetic analogs both functionalize an unreactive sp 3 -carbon and introduce an additional stereocenter into the molecule.…”

Mechanism of action of nucleoside antibacterial natural product antibiotics

“…The unusual amino acid meta-tyrosine is 6 biosynthesised from L-Phe by a novel non-heme iron-and tetrahydrobiopterin-dependent hydroxylase [32]. Mutasynthesis has been used to generate novel chlorinated pacidamycin derivatives [33], and modified sansanmycins [34]. The modified sansanmycins were reported to retain antimicrobial activity, in some cases with reduced activity, but MX-6 containing 4fluorophenylalanine at the C-terminus showed enhanced antimicrobial activity against B. subtilis and M. tuberculosis [34].…”