Improving the N-terminal diversity of sansanmycin through mutasynthesis
© Shi et al. 2016
Received: 3 February 2016
Accepted: 24 April 2016
Published: 6 May 2016
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.
Keywordsm-Tyr ssaX deletion mutant Mutasynthesis Novel sansanmycin analogues
Recently, the biosynthetic gene clusters for pacidamycins [5, 9], napsamycins , and sansanmycins  were identified and characterized, indicating that the assembly of the pseudo-tetrapeptide chain is catalyzed by nonribosomal peptide synthetases (NRPSs) with highly dissociated modules . Besides, the biosynthesis of uridyl pentapeptide of pacidamycins was catalyzed by the tRNA-dependent aminoacyltransferase PacB, which transferred the alanyl residue from alanyl-tRNA to the N-terminus of the pseudo-tetrapeptide . In contrast to ribosomal peptide synthesis, non-ribosomally assembled peptides contain not only the 20 proteinogenic amino acids but also many different building blocks, such as DABA, D-amino acids, hydroxyl amino acids, N- and C-methylated amino acids etc. Among them, non-proteinogenic amino acid meta-tyrosine (m-Tyr) is rarely found in bacterial secondary metabolites. UPAs and a potent cyclophilin inhibitor sanglifehrin A (SFA) are two examples containing m-Tyr as one of the building blocks. SfaA, identified in the SFA biosynthetic gene cluster, was speculated to catalyze the biosynthesis of m-Tyr . As a homologue of SfaA in pacidamycin biosynthetic gene cluster, PacX was characterized as a phenylalanine 3-hydroxylase that catalyzed the synthesis of m-Tyr from L-phenylalanine (L-Phe) in vitro . In sansanmycin biosynthetic gene cluster, SsaX is homologous to PacX with amino acid identity of 80 % across the whole protein, indicating that it is responsible for the biosynthesis of m-Tyr in Streptomyces sp. SS.
Although natural UPAs have potential to treat refractory infections, there is no UPAs entering clinical trials until now mainly due to their relatively poor physicochemical property. In previous studies, the N-terminal amino acid of the tetrapeptide of UPAs was supposed to be important functional group for the inhibition of MraY [16, 17]. It was proposed that the protonated ammonium ion binds in place of the Mg2+ cofactor at the MraY active site via cis-amide linkage . The N-terminal amino acid of known UPAs is almost dominated by m-Tyr or different bicyclic acids (possibly derived from m-Tyr), except that some pacidamycins possess Ala instead (Fig. 1). In this study, we focus on the substitution of the N-terminal amino acid to get novel sansanmycin analogues by mutasynthesis. Mutasynthesis is a useful method in the generation of new antibiotic derivatives . This approach could expand the chemical diversity of secondary metabolites and produce novel compounds with improved physicochemical properties or altered bioactivity. For example, it has been successfully employed to get novel nucleoside antibiotics such as nikkomycin analogues  and new ansamycin derivatives . However, mutational biosynthesis has not been employed to obtain UPA derivatives so far.
Here, we demonstrated that SsaX is responsible for the biosynthesis of m-Tyr in vivo by gene deletion and complementation and the sansanmycin production could be increased through the overexpression of ssaX. Six new sansanmycin analogues were purified and characterized in ssaX deletion mutant, indicating the substrate flexibility of the responsible NRPS. To expand the diversity of sansanmycins by mutasynthesis, different types of substrates were fed to the ssaX deletion mutant and some novel sansanmycin derivatives were obtained. These compounds were purified and structurally identified, some of which exhibited improved antibacterial activity or stability.
In-frame deletion of ssaX and its complementation
To further investigate the role of ssaX in sansanmycin biosynthesis, the plasmid pL-ssaX was transferred into the wild type strain by conjugation to give the ssaX overexpression strain SS/pL-ssaX, with pSET152 transferred strain SS/pSET152 as a control. With the same growth curves, the overexpression of SsaX apparently increased the production of sansanmycin A by 20 % from HPLC analysis (Fig. 2d). The antibacterial activity against P. aeruginosa 11 also showed that SS/pL-ssaX exhibited bigger inhibition zone than the control strain (Fig. 2e). This result, together with the result of chemical complementation, suggested that the biosynthesis of m-Tyr catalyzed by SsaX is at least one of the rate-limiting steps in the sansanmycin production .
Isolation and structural determination of sansanmycin analogues in ssaX deletion mutant
Through the HPLC and LC/MS analysis, a series of minor components of sansanmycin analogues were detected in the cultivated broth of ssaX deletion mutant SS/XKO. In order to characterize these compounds, SS/XKO fermentations were scaled up to obtain enough amount of material for further analysis. The target compounds were enriched by macroporous absorbant resin from fermentation broth, then isolated using DEAE-Sephadex A25 guided by HPLC–UV to yield the crude sansanmycin analogues. Subsequently, the crude compounds were purified by preparative HPLC. As a result, six new sansanmycin analogues were obtained and designated as sansanmycin MX-1 to MX-6 respectively. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS and nuclear magnetic resonance (NMR) spectroscopic analysis.
MX-3 has a molecular weight of 824, 16 mass units smaller than that of sansanmycin H , attributed to the loss of an oxygen atom. Furthermore, the ESI-MS/MS spectrum of MX-3 also showed the same diagnostic fragment with sansanmycin H, m/z 678 for the loss of the N-terminal m-Tyr, which suggested that the m-Tyr in sansanmycin H was replaced by a Phe in MX-3. Comparison with that of sansanmycin H, the 1H NMR spectrum of MX-3 (Additional file 1: Figure S10) showed a different aromatic pattern from sansanmycin H, with a Phe [δ 7.15 (m, 2H), 7.15 (m, 2H), 7.27 (m, 1H)] instead of m-Tyr [δ 7.23 (t, 1H), 6.78 (d, 1H), 6.75 (d, 1H), 6.72 (s, 1H)] .
MX-5 has a molecular weight of 863, 16 mass units greater than MX-4, corresponding to an extra oxygen atom. Comparison with that of MX-4, the 1H NMR spectrum of MX-5 (Additional file 1: Figure S11) showed a downfield shifted methyl proton signal [from δ 2.01 (−SCH3) to 2.46 (−SOCH3)], which hinted the oxidation of MX-4 to MX-5. The ESI-MS/MS analysis (Fig. 4) further confirmed this hypothesis.
The obtained six compounds MX-1–6 were new members of sansanmycin family. Compared with SS-A, MX-1, short of the N-terminal m-Tyr, bears a tri-pseudopeptide backbone that was found in the family of UPAs for the first time. It is the accumulated precursor when there is no m-Tyr present in SS/XKO, which is also the obvious evidence that SsaX catalyzes the biosynthesis of m-Tyr. The other five compounds were different from the known sansanmycins by the virtue of bearing Tyr, Phe and Met at the N-terminus, which were firstly reported in the family of UPAs. The presence of the new sansanmycin analogues with various N-terminal amino acids hinted that the NRPS responsible for the incorporation of the N-terminal amino acid into the tetra-pseudopeptide backbone has moderate substrate promiscuity, suggesting that certain amounts of sansanmycin analogues might be able to be generated by mutational biosynthesis using ssaX deletion mutant SS/XKO.
Generation of structurally diverse sansanmycin analogues using ssaX deletion mutant
Initially twenty proteinogenic amino acids including Phe, Tyr and Met were used to probe the feasibility of mutasynthesis. The production of sansanmycin MX-2 which bears Tyr as its N-terminus was nearly doubled when fed SS/XKO with Tyr (3 mM) (Fig. 3). Similarly, the production of sansanmycin MX-4 which bears Phe as its N-terminus was increased to two to three times when fed with Phe (3 mM) (Fig. 3). But the HPLC profile of the fermentation broth of ssaX deletion mutant fed with Met, as well as other proteinogenic amino acids (3 mM) had no obvious changes (data not shown). This may be explained by the substrate preference of the NRPS, which preferred to select Phe or Tyr rather than any other proteinogenic amino acids. This result is consistent with the production level of sansanmycin analogues in SS/XKO. The improved production of sansanmycin MX-2 and MX-4 by feeding substrates Phe and Tyr suggested that mutasynthesis might be suitable to produce sansanmycin analogues with alternate N-terminal substrate.
Comparison of the relatively production level of sansanmycin analogues in ssaX deletion mutant SS/XKO
Fed substrate (3 mM)
compound 1, 2
Antibacterial activity and stability of novel sansanmycin analogues
Activities of sansanmycin analogues
E. coli ΔtolC
P. aeruginosa 11
B. subtilis CMCC (B) 63501
During the past decade, considerable efforts have been made to exploit new UPA derivatives. Seventeen sansanmycin analogues were semi-synthesized with sansanmycin A as the starting material, but most of them exhibited less anti-mycobacterial activity in comparison with parent natural product . Strategy of precursor-directed biosynthesis was employed to get pacidamycin analogues with modified C-terminal amino acid through feeding Trp derivatives . The same strategy was also applied to sansanmycin-producing strain, resulting sansanmycin analogues with the C-terminus substituted by Phe derivatives . Although some UPA analogues were produced, few of them had significantly improved antibacterial activity and/or physicochemical property. In the past 5 years, the biosynthetic pathways have been studied extensively at the genetic, enzymatic and regulatory levels [9–13], and bioengineering approaches are available to be used in producing novel UPA derivatives. In this work, mutational biosynthesis is employed by blocking the biosynthesis of m-Tyr and then feeding variety of alternative substrates to produce novel sansanmycin derivatives. This strategy is efficient to obtain novel sansanmycin analogues, creating a great structural diversity at the N-terminus.
In most of the reported UPAs, the N-terminus of the tetra-pseudopeptide (AA1) was occupied by m-Tyr and its related bicyclic acids, except that pacidamycin D and S have an Ala at the N-terminus (Fig. 1). Two NRPSs responsible for selection of amino acid to incorporate into the N-terminus of pacidamycin have been reported. PacU was demonstrated to specifically activate Ala , and PacW was identified to activate m-Tyr . In sansanmycin-producing strain, there are also two homologues of PacU, SsaU and SsaW, existed in sansanmycin biosynthetic gene cluster , but their amino acid sequences were almost exactly the same, with only one alteration of Lys to Arg . Though they exhibited high overall homology with both PacU and PacW, the main residues in the binding pocket (specificity-conferring code) in the adenylation (A) domain [31, 32] were same with PacW but different from PacU. This is consistent with the fact that m-Tyr is the optimum substrate for the production of sansanmycin A in the wild type strain, and all the natural sansanmycin derivatives have m-Tyr and its related bicyclic acids at the N-terminus. When the biosynthesis of m-Tyr is blocked, Phe, Tyr, and small amount of Met may incorporate into the polypeptide of sansanmycin in ssaX deletion mutant, showing the potential of this mutant as a cell factory to expand the chemical diversity of AA1.
Various substrates including Phe, Tyr, halogenated Phe and other non-proteinogenic amino acids were fed to the ssaX deletion mutant and about 20 novel sansanmycin analogues with different N-termini were produced according to the substrate promiscuity of the A domain of SsaU and SsaW. Ten of these compounds were purified and structurally determined by ESI-MS, ESI-MS/MS (Fig. 4) and NMR. According to the production level of the compounds in ssaX deletion mutant fed with corresponding substrate (Table 1), the preference of the A domain in charge of activating the sansanmycin N-terminal amino acid is m-Tyr > Phe ≥ ortho-halogenated phenylalanines ≥ 2-furylalanine > Tyr > 2-thienylalanine ≥ p-amino-phenylalanine > Met. Out of our expectation, 2-furylalanine and 2-thienylalanine could be incorporated into the sansanmycin, suggesting the diversity of AA1 might be worth to explore further by trying more structurally diverse substrates. The compounds produced by mutasynthesis in this study retained the anti-TB activity of sansanmycin A in vitro, and more encouragingly, they showed similar activity to MDR and even XDR strains isolated from patients. Meanwhile, the stability of MX-2 and MX-4 was demonstrated to be greatly improved compared to sansanmycin A.
From the result of previous researches on the UPA derivatives of the wild type strains [8, 25] or by precursor-directed biosynthesis [26, 28], the A domain responsible for activating the C-terminal amino acid (AA4) has relatively high substrate promiscuity from Trp, Tyr to Phe and substituted Phe and Trp. On the other hand, the amino acid at the position of AA3 also can vary from Met and Ala  to Leu and Phe in sansanmycins [25, 33]. Now, together with the diversity at AA1 produced in this study, the combination of the variations in these three parts of the polypeptide backbone could be potentially expected that hundreds of new sansanmycin analogues might be obtained. Recently, crystal structure of Aquifex aeolicus MraY has been published , and the residues (Asp117, Asp118, Asp265, and His324) important for the activity of MraY in the active site have been elucidated . The structural information of MraY from A. aeolicus sets foundations for homologous modeling of MraY from M. tuberculosis [34, 35], which will facilitate the study on structure activity relationship (SAR) of novel chemically diverse UPA derivatives obtained by further rationale genetic engineering manipulation.
It is demonstrated that SsaX is responsible for the biosynthesis of m-Tyr in vivo by gene deletion and complementation and the sansanmycin production could be increased through the overexpression of ssaX. Six new sansanmycin analogues were purified and characterized in ssaX deletion mutant, indicating the substrate flexibility of the responsible NRPS. The diversity of sansanmycin was further expanded by mutasynthesis, in which different types of substrates were fed to the culture of ssaX deletion mutant. Totally ten compounds were purified, structurally identified and firstly reported. Five of them displayed anti-mycobacterial activity comparable to sansanmycin A and especially, they are active to MDR and even XDR M. tuberculosis clinical strains. In addition, sansanmycin MX-2 and MX-4 displayed significantly improved stability than sansanmycin A. These improved properties may promote the novel anti-TB drug investigation targeting a clinically unexploited target MraY.
Strains, plasmids and growth conditions
Strains and plasmids used in this study
Streptomyces sp. SS
Wild-type strain (sansanmycin-producing strain), CPCC 200442
Mutant of Streptomyces sp. SS with the in-frame deletion of ssaX
SS/XKO with the expression vector pL-ssaX, Amr
Streptomyces sp. SS with the expression vector pL-ssaX, Amr
General cloning host
Donor strain for intergeneric conjugation between E. coli and Streptomyces, Cmr, Kmr
Strain for RED/ET-mediated recombination, Cmr
Strain for testing antimicrobial activity
Pseudomonas aeruginosa 11
Strain for sansanmycin bioassays
Bacillus subtilis CMCC (B) 63501
Strain for testing antimicrobial activity
Strain for testing antimicrobial activity
Standard strain, susceptible to isoniazid and rifampicin
Clinically isolated multi-drug-resistant strain, resistant to isoniazid and rifampicin
Clinically isolated multi-drug-resistant strain, resistant to isoniazid and rifampicin
Clinically isolated extensive-drug-resistant strain, resistant to isoniazid, rifampicin, ethambutol, streptomycin, kanamycin and ofloxacin
Cosmid based on vector pOJ446, containing the majority of sansanmycin biosynthetic gene cluster ssaM–ssaV including ssaX
Cosmid 13R-1with the minimal replicon of SCP2* replaced by ampicillin resistance marker bla, Ampr, Amr
Cosmid 13R-1-SCP2KO with the in-frame deletion of ssaX, Ampr, Amr
Vector used as the template for amplifying aadA cassette, Specr
Streptomyces integrative vector, Amr
pSET152 derivative containing the constitutive promoter ermE*p, Amr
pL646 derivative plasmid containing 843 bp complete coding region of ssaX
Construction and complementation of Streptomyces sp. SS ssaX mutant
The ssaX in-frame deletion mutant SS/XKO was constructed by the λ-RED mediated PCR targeting method , using cosmid 13R-1  covering ssaM–ssaV of sansanmycin biosynthetic gene cluster. In order to disrupt ssaX through homologous recombination, the minimal replicon of SCP2* of 13R-1 was firstly replaced by ampicillin resistance marker bla, resulting 13R-1-SCP2KO. Then, a streptomycin resistance cassette (aadA gene) was amplified with primers PT-X-1 (5′-GCGGGAGGCCCCGCTGAACAGGGCCGCGATGCTGTCGTCATTCCGGGGATCCGTCGACC-3′) and PT-X-2 (5′-GTCACCGACACCGCCTATGAGAAGCGCCGCGAGGAGATCTGTAGGCTGGAGCTGCTTC-3′) including two 39-nt homologous extensions to sequences up- and downstream of the target ssaX gene. The cassette was introduced into E. coli BW25113/pIJ790 to substitute ssaX on cosmid 13R-1-SCP2KO. The streptomycin resistance cassette on the correct recombinant cosmid was removed by FLP-recombinase in E. coli DH5α/BT340. The mutant cosmid 13R-1-SCP2KO-XKO was introduced into E. coli ET12567/pUZ8002 and then transferred into Streptomyces sp. SS by conjugation. Double-crossover exconjugants (Ams) were selected on MS agar with and without Am and confirmed by PCR using primers PT-X-7 (5′-TGAAGCCCGCCGCCTTTC-3′) and PT-X-8 (5′-TCTGCCTTCCGCCTGACCAT-3′) and southern blot hybridization using DIG Prime DNA Labeling and Detection Starter Kit I (for color detection with NBT/BCIP, Roche). The genomic DNAs were digested with BamHI and hybridized with specific probes of ssaX deleted fragment amplified with primers SB-X-1 (5′-CTCGACCTCGTTCATGGAGT-3′) and SB-X-2 (5′-AGTACGTCGACTGGGAGCAC-3′) and the fragment downstream of ssaX amplified with primers SB-X-3 (5′-AGAAACCACGATGCGAAATC-3′) and SB-X-4 (5′-TGGATTTTTCGCTTCAAACC-3′) respectively. The resulted ssaX deletion mutant was designated SS/XKO.
For complementation analysis, complete ssaX coding region was amplified using primers pL-ssaX-F (5′-CGCATATGCAAGGGCATCGCGAC-3′) and pL-ssaX-R (5′-ATAGGATCCTCAGCGCCGGGTGCC-3′), and then cloned into the NdeI and BamHI sites of a pSET152-derived expression plasmid, pL646 , under the control of a strong constitutive promoter ermE * p. The resulted expression vector pL-ssaX was transferred into SS/XKO and Streptomyces sp. SS by conjugation to give the complementation strain and ssaX overexpression strain respectively. The plasmid pSET152  was transferred to SS/XKO and the wild type strain respectively as controls.
Analysis of sansanmycin production
Fermentation, isolation, and high-pressure liquid chromatography (HPLC) analysis of sansanmycins were carried out as described previously [1, 25]. In brief, pieces of well-grown agar cultures of different strains were firstly inoculated in fermentation medium and cultured at 28 °C for 48 h at 200 rpm. The obtained seed cultures were trans-inoculated into three parallel 100 ml fermentation medium by 5 % inoculation and grown at 28 °C for 5 days at 200 rpm. In the feeding test, each exogenous substrate was added to the fermentation medium to the final concentration of 3 mM. At indicated time points, five-milliliter cell cultures were collected by centrifugation and dried at 60 °C to constant weight for monitoring the growth curve. The obtained supernatants were analyzed for antibacterial activity and production of sansanmycins by bioassay and HPLC. Antibacterial activity was measured by cylinder plate method using P. aeruginosa 11. For analyzing the expected analogues, the supernatant of fermentation broth was enriched by Sep-Pak C18 Classic Cartridge (Waters Associates, Milford, MA, USA), eluted with 60 % methanol solution. The effluent was subjected to HPLC on an XBridge™ C18 column (4.6 × 150 mm, 3.5 μm, Waters, Dublin, Ireland) maintained at 40 °C, with a gradient of 80:20 0.1 % (w/v) (NH4)2CO3-MeOH to 40:60 in 40 min as mobile phase at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm. For the analysis of sansanmycin MX-3 and MX-6, the mobile phase was changed to 10:90 MeOH-H2O (pH adjusted to 12.0 with NH3·H2O) in 40 min.
Purification of sansanmycin analogues
Isolation and purification of sansanmycin analogues was performed following the method of Xie et al.  with some modifications. Fifty liters of fermentation supernatant was obtained by centrifugation and then applied on a column of macroporous absorbant resin 4006. The active materials were eluted with 30 % aqueous acetone. Then the effluent was applied on Toyopearl DEAE-Sephadex A25 eluted with Tris-HCl (20 mM, pH 8.5) plus NaCl and monitored by HPLC-UV. The concentration of NaCl was adjusted with different compounds from 0.01 to 0.05 M. The effluent containing target compounds was collected and further purified by preparative HPLC (YMC-Pack ODS-A 5 μm, 250 × 20 mm column, 0.1 % (w/v) (NH4)2CO3-MeOH; flow rate, 5 ml/min; UV detection at 254 nm and oven temperature at 40 °C). The ratio of 0.1 % (w/v) (NH4)2CO3 and MeOH was dependent on different compounds. The structures of obtained compounds were determined using ESI-MS and ESI-MS/MS (ThermoFisher LTQ Orbitrap XL mass spectrometer) as well as NMR [Varian Mercury 600 spectrometers, in dimethyl sulfoxide (DMSO)-d 6 ].
The minimum inhibitory concentrations (MICs) for M. tuberculosis strains were determined by the microplate Alamar blue assay (MABA) . All M. tuberculosis strains were grown on Middlebrook 7H9 medium supplemented with 0.2 % (v/v) glycerol and 10 % (v/v) OADC (oleic acid, albumin, dextrose, catalase) until the mid-log phase of growth at 37 °C. The final suspension of bacteria cells were diluted in Middlebrook 7H9 medium to 106 cfu/ml. Initial compound dilutions were prepared in DMSO, and subsequent twofold dilutions were performed in 100 μl of 7H9 (no Tween 80) in the microplates. Then, the MIC was measured in sterile 96-well plates with 100 μl of the bacterial suspension and 100 μl compound dilution per well. The MIC was defined as the lowest concentration of drug that prevented the color change of Alamar blue reagent from blue to pink. Rifampicin, isoniazid, ethambutol and streptomycin were used as controls.
The MICs for other bacterial strains were determined by a microdilution test following recommendations from the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) . The bacterial strains were grown on Mueller–Hinton broth (MHB) , and the final suspension of bacteria (in MHB medium) was adjusted to 106 cells/ml. The dilutions of tested compounds were performed as method above with MHB medium instead. Then serial dilutions (100 μl) were transferred to a 96-well plate in triplicate, and 100 μl of the bacterial suspension was added to each well. After incubation at 37 °C for 24 h, the MIC was defined as the lowest concentration that inhibited the growth of the tested organism detected by visual observation. Streptomycin was used as the positive control.
Stability determination of sansanmycin analogues
To dissect the stability of sansanmycin A and other sansanmycin analogues, compounds were dissolved in 0.05 M KH2PO4 buffer (pH adjusted to 6.0 with NaOH). All samples were incubated at 25 °C for 9 days. Each sample has three parallel repeats. Residual analogues were analyzed by HPLC and quantified by the peak areas.
YS carried out experiments, analyzed the primary data and wrote the draft manuscript. ZJ and XL assisted with feeding experiments. NZ assisted with data analysis of MS and NMR, QC assisted with data analysis of MS, and QL designed ssaX mutant and assisted with relevant experiments. LW and SS assisted with testing antibacterial activity. YX supervised the chemical work in this study and revised the manuscript. BH supervised the whole research work and revised the manuscript. All authors read and approved the final manuscript.
We thank Dr. Bertolt Gust (Pharmaceutical Institute, University of Tuebingen, Germany) for kindly providing Escherichia coli ΔtolC mutant strain. We thank Professor Kanglin Wan from the Chinese Center for Disease Control and Prevention (CCDC) for testing the anti-TB activity of compounds. This work was supported by the National Mega-Project for Innovative Drugs (2015ZX09102007-016, 2012ZX09301002-001-016 and 2014ZX09201001-004-001) and the National Natural Science Foundation of China (81321004, 81402836, 81273415, 81302677 and 31170042).
The authors declare that they have no competing interests.
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