Discovery of New Antibacterial Accramycins from a Genetic Variant of the Soil Bacterium, Streptomyces sp. MA37

Continued mining of natural products from the strain Streptomyces sp. MA37 in our laboratory led to the discovery of a minor specialized metabolite (SM) called accramycin A. Owing to its low yield (0.2 mg/L) in the wild type strain, we investigated the roles of regulatory genes in the corresponding biosynthetic gene cluster (acc BGC) through gene inactivation with the aim of improving the titer of this compound. One of the resulting mutants (∆accJ) dramatically upregulated the production of accramycin A 1 by 330-fold (66 mg/L). Furthermore, ten new metabolites, accramycins B–K 2–11, were discovered, together with two known compounds, naphthacemycin B1 12 and fasamycin C 13 from the mutant extract. This suggested that accJ, annotated as multiple antibiotic resistance regulator (MarR), is a negative regulator gene in the accramycin biosynthesis. Compounds 1–13 inhibited the Gram-positive pathogens (Staphylococcus aureus, Enterococcus faecalis) and clinical isolates Enterococcus faecium (K59-68 and K60-39) and Staphylococcus haemolyticus with minimal inhibitory concentration (MIC) values in the range of 1.5–12.5 µg/mL. Remarkably, compounds 1–13 displayed superior activity against K60-39 (MIC = 3.1–6.3 µg/mL) compared to ampicillin (MIC = 25 µg/mL), and offered promising potential for the development of accramycin-based antibiotics that target multidrug-resistant Enterococcus clinical isolates. Our results highlight the importance of identifying the roles of regulatory genes in natural product discovery.

Based on the previous knowledge of fasamycins and formicamycins [4], the putative biosynthetic gene cluster (acc BGC) of 1 was identified [25]. The biosynthetic enzymes encoded in the acc BGC display high amino acid (AA) similarity with the ones in fasamycins and formicamycins, including the FAD-dependent halogenation enzyme (AccV) which shares high AA identity with the chlorinase, ForV, encoded in the fasamycin and formicamycin BGCs from Streptomyces formicae [4]. Unlike S. formicae, which produces an array of mono-, di-, and tri-chlorinated fasamycins and formicamycins, the MA37 WT strain appears to be less productive in terms of accramycin yield and chemical diversity, suggesting poor expression of the acc BGC in the MA37 WT strain [25]. It was hypothesized that this may be due to the presence of negative regulatory genes that suppress the biosynthesis of accramycins.
Herein, we report the hyper accramycin producer of an MA37 variant by inactivating the putative regulatory gene (accJ), which encodes for the multiple antibiotic resistance regulator (MarR). Subsequent chemical workup and structural elucidation allowed for the discovery of more than ten new accramycin congeners, together with two known compounds, naphthacemycin B 1 12 [26] and fasamycin C 13 [4]. Except for compounds 3 and 4, compounds 5-11 contain multi-chlorines installed at various positions of the accramycin scaffold, suggesting that the putative enzyme (AccV) is a promiscuous chlorinase. A similar observation was observed for the deletion of the forJ gene (accJ homologue) in S. formicae, which also resulted in improved titer of fasamycins and formicamycins [27], in parallel to our study.

General Experimental Procedures
Agilent 1260 Infinity (Scotland, UK) was used for high pressure liquid chromatography (HPLC) separation. IR spectra were obtained using a PerkinElmer Spectrum version 10.4.00 Fourier transform infrared (FTIR) spectrometer (2013) (Scotland, UK) equipped with an attenuated total reflection (ATR) diamond cell. HR-ESIMS were determined on an LC-MS Thermal Science Mass Spectrometry (LTQ Orbitrap, Scotland, UK) coupled with a thermal instrument HPLC (Accela PDA detector, Accela PDA autosampler and Accela Pump, C18 Sunfire 4.6 × 150 mm waters, Scotland, UK). The NMR spectra were recorded on a Bruker AVANCE III HD 400 MHz (Ascend™ 9.4 Tesla) and/or Bruker AVANCE III HD 600 MHz (Ascend™ 14.1 Tesla, Scotland, UK) with Prodigy TCITM cryoprobe.

Strain, Genomic DNA, and Media
The Streptomyces sp. MA37 strain was isolated from the soil sample collected from Legon, Ghana, Africa [20]. Genomic DNA from MA37 was extracted as described previously [24]. All media/broth for fermentation, solvents, and chemicals were obtained from Fisher Scientific (Scotland, UK), unless otherwise stated.

Conjugations between Escherichia coli and Streptomyces sp. MA37
The confirmed deletion construct was introduced to Escherichia coli S17-1 by heat-shock (42 • C, 1 min), and the resulting strains were used as conjugal donors to MA37. The MA37 mycelia were used for conjugation as the strain produces no spores, and were prepared as described previously [24]. The mycelia (0.5 mL) was mixed with E. coli S17-1 (0.5 mL), followed by centrifugation (5 min). The supernatant was discarded, and the remaining mixture was plated onto SFM (soya flour (4 g), mannitol (4 g), agar (4 g), in 200 mL H 2 O) containing MgCl 2 (10 mM). The plate was incubated at 28 • C. After 16-20 h, the plate was overlaid with water containing nalidixic acid (25 µg/mL, 1 mL) to inhibit the growth of E. coli and apramycin (50 µg/mL) for selection of successful exconjugants. Incubation was continued until the ex-conjugates appeared (28 • C, 3-5 days).

Screening for the Mutant Strain
The resulting ex-conjugants were colony-purified at 28 • C on ISP2 (glucose (4 g), yeast extract (4 g), malt extract (10 g), agar (20 g), in 1 L H 2 O) containing apramycin (50 µg/mL), and then re-streaked onto ISP2 with no antibiotic selection, and incubated at 37 • C for 1-3 generations to promote plasmid loss. Using the streak plate method, each single colony was inoculated separately onto ISP2 and ISP2 with apramycin (50 µg/mL). Strains sensitive to apramycin (indicating plasmid loss) were subsequently confirmed by PCR screening using internal primers. Glycerol stocks of the verified mutant strains (∆accF, ∆accJ, ∆accI, and ∆accP) were prepared and stored in an −80 • C freezer.
The cultures were incubated (7 days, 28 • C, 180 rpm), after which Diaion®HP-20 (3 g/50 mL solution) was added. Incubation was continued for the next 18-24 h (28 • C, 180 rpm). The resin was filtered, extracted exhaustively with methanol, followed by concentration using a rotary evaporator (Buchi Rotavapor R200, Scotland, UK). Likewise, the MA37 WT strain was fermented and extracted as in the mutant strains. The mutants and WT extracts were subjected to mass spectrometric and HPLC-UV analyses monitored at λ450 nm, and the chemical profiles of each were compared. Several peaks were observed in the ∆accJ extract with the characteristic accramycin UV pattern (226, 250, 286, 355, and 420 nm) not detected in the WT or other mutant strains.

Fermentation, Extraction, Metabolite Screening
Two-liter fermentation culture of the ∆accJ mutant strain was carried out in ISP2 broth. Four 2.0-L baffled flasks (Corning™ polycarbonate), each containing 500 mL ISP2, were inoculated with seed culture (1:100), and plugged with foam stoppers (Fisherbrand™ polyurethane). Fermentation, incubation, and extraction of the ∆accJ cultures were carried out as described above. The methanol extracts were combined, evaporated to dryness under reduced pressure to yield crude extract (7 g), which was subjected to high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis.
Accramycin   [28], and belonged to the complex clonal 17 sub-cluster, which is highly resistant to ampicillin [28,29]. S. haemolyticus 8-7A was obtained from the same hospital; all clinical strains were provided courtesy of Prof. Kristin Hegstad. The activity of 1-13 was determined using a sequential 2-fold serial dilution of the compounds (50-0.10 µg/mL) in DMSO following the standard protocols recommended by the Clinical and Laboratory Standard Institute [30] and as previously described [25,31,32]. The MIC was defined as the lowest concentration of the compound that inhibited ≥ 95% bacterial growth after overnight incubation. Ampicillin (Sigma) was used as the antibiotic standard.

GenBank Accession Number
The accramycin BGC was deposited in NCBI and was assigned the accession number MN477201.

Results and Discussion
Bioinformatics analysis suggested that the acc BGC encodes four putative pathway-specific regulators, including LuxR (AccF), MarR (AccJ), LysR (AccI), and MerR (AccP) transcriptional regulators (Table S1) [25]. To assess their roles in the production of accramycins, we carried out the in-frame deletion of these four genes, generating four MA37 variants, respectively. The resulting mutants were then cultivated in ISP2 (7 days) and subsequently extracted with methanol to yield four crude extracts. Among the four samples, only the extract from the ∆accJ variant displayed a significant metabolic profile in HPLC and high-resolution electrospray ionization mass spectrometry (HRESIMS) analyses compared with the WT ( Figure S2). Of particular relevance was the presence of several new HPLC peaks with the characteristic UV pattern (226, 250, 286, 355, and 420 nm), which had identical UV absorption to the one of accramycin A we previously isolated [25]. This finding was also supported by the more intense yellow color of the ∆accJ culture compared to the WT, which pointed to the yellow naphthacene chromophore [4,25].
To further confirm the identities of the newly emerged metabolites in the ∆accJ variant, we set out a large-scale fermentation (2 L) for chemical workup and structural elucidation. The crude extract was first fractionated through vacuum liquid chromatography to generate ten fractions. HPLC analysis of these ten fractions confirmed the presence of the accramycin constituents in fractions 3 (F3) and 4 (F4). The HPLC-UV-targeted isolation approach afforded accramycin A 1 in a significantly improved titer (66 mg/L), a 330-fold increase compared to the one from the WT (0.2 mg/L) [25]. Additionally, several new accramycin analogues 2-11, together with two known compounds naphthacemycin B 1 12 [26] and fasamycin C 13 [4], were isolated. Likewise, the titer of fasamycin C was enhanced in the mutant strain by 30-fold (26 mg/L).

Structure Elucidation
New accramycin analogues were obtained as yellowish to red powders. Inspection of the HRESIMS data and MS/MS fragmentation pattern indicated that compounds 1-2 are non-halogenated, 3-4 have one chlorine atom, 5-7 have two chlorine atoms, 8-9 are trichloro-substituted, and 10-11 are tetra-chlorinated. Thorough analyses of the UV, HRESIMS, and nuclear magnetic resonance (NMR) data of 1, 12, and 13 indicated that compounds 1, 12, and 13 are known metabolites, accramycin A [25], naphthacemycin B 1 produced by Streptomyces sp. KB-3346-5 ( Figures S3-S22) [1,26], and fasamycin C produced by S. formicae (Figures S21-S26) [4], respectively. The structures of accramycins B-K 2-11 ( Figure 2, Figures S23-S76) were elucidated by comparison of the observed UV, molecular formulae, and NMR data with the reported data of 1, 12, and 13 (Tables S2 and S3). The molecular formula C 28 H 23 ClO 7 of compound 3 deduced by HRESIMS had 34 mass units more than 13, which suggested that 3 was a mono-chlorinated analogue of 13. This was supported by the isotope pattern observed for 3 (m/z 507.1205, calcd [M+H] + : 509.1166) in a 3:1 ratio. The lower-field shifting of carbon signal (δ C-3 106.6) in ring A indicated that the electronegative chlorine atom was attached to C-3 in the structure. This was further supported by the HMBC correlation from H-1 (δ H 6.96) to C-3. The cross peak from H 3 -28 (δ H 3.80) to C-24 (δ C 159.0) established the connectivity of the methoxy functional group at C-24 in 3. The additional chlorine atom was assigned at C-3 based on the HMBC correlation from H-1 (δ H 6.97) to C-3 (δ C 106.3). Compounds E 5 and F 6 are isomers with the same molecular formula, C 29 H 24 Cl 2 O 7 deduced by HR ESIMS. The 68 Da difference of 5-6 from 1 and the isotope fragmentation pattern (m/z 555.0978: 557.0944: 559.0925) in 9:6:1 ratio supported the presence of two chlorine atoms in the formula. Two chlorines were placed at C-3 and C-13 in 5-6, based on the HMBC correlations from H-1 (δ H 6.87) to C-3 (δ C 106.8), H-11 (δ H 7.05) to C-13 (δ C 115.2), and H-1 (δ H 7.00) to C-3 (δ C 107.6), H-11 (δ H 6.87) to C-13 (δ C 112.9), respectively. They differed in the attachment of the two methoxy groups, where one was attached to C-24 in 5-6, while the other was linked to C-12 in 5 and C-2 in 6. This was confirmed by the correlations from H 3 -29 (δ H 4.02) to C-12 (δ C 155.8) and from H 3 -30 (δ H 4.06) to C-2 (δ C 161.5) in the HMBC spectra of 5 and 6, respectively.
The 1 H and 13 C NMR spectra of compound 7 were similar to those of 6 except for ring D. The molecular formula of 7 C 30 H 26 Cl 2 O 7 showed 14 mass units more than 6, indicative of an additional methyl group in the structure which was assigned at C-12 based on the HMBC correlations from H 3 -29 (δ H 4.02) to C-12 (δ C 156.6).
Compound 8 is a tri-chlorinated isomer with the molecular formula, C 28 H 21 Cl 3 O 7 deduced by HRESIMS. The 1 H-NMR spectrum of 8 revealed only one methoxy group which was assigned at C-2 based on the HMBC cross-peaks between H 3 -30 (δ H 4.05) and C-2 (δ C 161.3). The three chlorines were designated based on the downfield carbon shifts observed in C-3 (δ C 108.8), C-23 (δ C 107.5), and C-25 (δ C 113.2) in comparison with non-chlorinated 12, the attachment of which was further supported by HMBC correlations from H-1 (δ H 6.97) to C-3, H 3 13 C NMR spectra of compound 10 were similar to 8 except for ring D. HRESIMS analysis of 10 showed 34 mass units more than 8, indicating an additional chlorine substituent in the structure, which was assigned at C-13 based on the HMBC correlation from H-11 (δ H 6.87) to C-13 (δ C 113.8).
The 1 H-NMR spectrum of compound 11 showed an additional singlet signal in the methoxy region compared to 10. The HMBC cross peak between H 3 -29 (δ H 4.02) and C-12 (δ C 156.2) designated the methoxy group at C-12 of ring D in the structure.
On the basis of the evidence in this study, compounds 2-11 were confirmed as new members of accramycin polyketides for which the names accramycin B-K are proposed, respectively.  Table 1). The presence of multiple chlorines at rings A, D, or E did not enhance the activity as observed in accramycins C-K consistent with previous findings [7]. The O-methyl-bearing accramycin B exhibited slightly higher MIC against all the tested pathogens. Conversely, the presence of free hydroxyl groups at C-12 and C-24 favored antibacterial activity as observed in accramycin J. Compound 10 in comparison with naphthacemycin B 1 supported that the O-methyl at C-2 is a preferred structural feature for the activity. Furthermore, accramycin J (MIC = 6.3 µg/mL) exhibited 2-fold inhibitory activity over ampicillin (MIC = 12.5 µg/mL) against the clinical isolate, S. haemolyticus. Notably, compounds 1-13 had superior activity (MIC = 3.1-6.3 µg/mL) against E. faecium K60-39 than ampicillin (MIC = 25 µg/mL). Thus, the accramycins represent potential therapeutic lead molecules for the development of potent drugs against ampicillin-resistant Enterococcus clinical strains. None of the compounds 1-13 displayed activity against the Gram-negative pathogens, E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853), and the fungal pathogen C. albicans (ATCC 10231) at the highest concentration tested (50 µg/mL).

Conclusions
In conclusion, we confirmed that accJ is a repressor gene in accramycin biosynthesis. Inactivation of the accJ gene resulted in the production of thirteen fasamycin-type antibiotics in Streptomyces sp. MA37 ∆accJ mutant including accramycins A-K 1-11 along with two known compounds, naphthacemycin B 1 12 and fasamycin C 13. The structures of compounds 1-13 were deduced by HRESIMS and 1D and 2D NMR. The fermentation titers of the isolated metabolites were significantly improved, particularly accramycin A with an estimated yield from 0.2 mg/L (WT strain) to 66 mg/L (∆accJ mutant strain). During the preparation of this manuscript, Devine et al. reported a similar observation in the producer of fasamycins and formicamycins, S. formicae, suggesting that inactivation of forJ, encoding the MarR regulator (homologue to AccJ), resulted in an improved titer of fasamycins and formicamycins [27]. Six new fasamycin analogues L-Q and two new formicamycins R-S were characterized in this variant [27]. Compounds 1-13 exhibited good activity against the Gram-positive pathogens, S. aureus and E. faecalis, and clinical isolates, E. faecium K59-68, E. faecium K60-39, and S. haemolyticus (MIC = 1.5-12.5 µg/mL). Remarkably, compounds 1-13 displayed better inhibitory activity over ampicillin against K60-39. Hence, the accramycin pharmacophore represents potential lead molecules for the development of potent antibiotics that target Enterococcus isolates.