Page S9: Figure S1: Scores and Loadings Plot for the Identification of Bottromycin D (1) .... constructed using Tree Builder at the Ribosomal Database Project site ...
Supporting Information
Structure and Biosynthesis of the Antibiotic Bottromycin D Yanpeng Hou,† Ma. Diarey B. Tianero,‡ Jason C. Kwan, ‡ Thomas P. Wyche, † Cole R. Michel, † Gregory A. Ellis, † Emmanuel Vazquez-Rivera, † Doug R. Braun, † Warren Rose, † Eric W. Schmidt, ‡ and Tim S. Bugni*,† Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States, Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, 84112, United States Table of Contents: Page S1-S9:
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Experimental Section General Experimental Procedures Fermentation and Isolation of Bottromycin D (1) Fermentation and Isolation of Bottromycin A2 Marfey’s Analysis of Bottromycin D (1) Whole Genome Sequencing and Biosynthesis Figure S1: Scores and Loadings Plot for the Identification of Bottromycin D (1) Figure S2: Schematic of the Contigs from Genome Sequencing Table S1: NMR Data for 1, Conformer 1 Table S2: NMR Data for 1, Conformer 2 Table S3: Primers used in This Study Figure S3: Nucleotide Sequence and Precursor Peptide Sequence Figure S4: LCMS of the Transformant Ala3-Val (pSET-bstA1) to Produce Bottromycin A2. Figure S5: Accurate Mass Measurements for Bottromycin A1 and Bottromycin D (1). References for Experimental Procedures Figure S6: 1H NMR Spectrum of 1 at 600 MHz in CDCl3 Figure S7: 1H NMR Spectrum of 1 at 500 MHz in DMSO-d6/CD3CN Figure S8: 13C NMR Spectrum of 1 at 125 MHz in CDCl3 Figure S9: COSY Spectrum of 1 at 600 MHz in CDCl3 Figure S10: HSQC Spectrum of 1 at 600 MHz in CDCl3 Figure S11: HMBC Spectrum of 1 at 600 MHz in CDCl3 Figure S12: ROESY Spectrum of 1 at 600 MHz in CDCl3 Figure S13: 1H-15N HSQC Spectrum of 1 at 600 MHz in CDCl3 Figure S14: Conformer Studies Figure S15: Conformer Studies
S1 EXPERIMENTAL SECTION General Experimental Procedures Optical rotations were measured on a Perkin–Elmer 241 Polarimeter. UV spectra were recorded on an Aminco/OLIS UV-Vis Spectrophotometer. IR spectra were measured with a Bruker Equinox 55/S FT–IR Spectrophotometer. NMR spectra were obtained in CDCl3 with a Bruker Avance 600 MHz spectrometer equipped with a 1.7 mm 1H{13C/15N} cryoprobe and a Bruker Avance 500 MHz spectrometer equipped with a 13C/15N{1H} cryoprobe. HRMS data were acquired with a Bruker MaXis 4G QTOF mass spectrometer. HR-LCMS data for the transformants were acquired on a Micromass Q-tof Micro and with a Waters 2795 HT HPLC system. RP HPLC was performed using a Shimadzu Prominence HPLC system and a Phenomenex Luna C18 column (250 × 10 mm, 5 µm). The Advanced Marfey’s method utilized a Waters Acquity UPLC coupled with a Bruker MaXis 4G mass spectrometer. Biological Material. Didemnum psammathode were collected in November 2010 in the Florida Keys (24° 47.976”, 81° 28.353”). For cultivation, a sample of ascidian (1 cm3) was rinsed with sterile seawater, macerated using a sterile pestle in a micro-centrifuge tube, and dilutions were made in sterile seawater, with vortexing between steps to separate bacteria from heavier tissues. Dilutions were separately plated on three distinct media: R2A1, ISP2 supplemented with artificial seawater2 and M4.3 Media were supplemented with 50 µg/ml cycloheximide and 25 µg/ml nalidixic acid. Plates were incubated at 29 °C, and WMMB272 was purified from M4. Fermentation, Extraction, and Isolation. Two 10 ml seed cultures (25 × 150 mm tubes) in medium ASW-A (20 g soluble starch, 10 g glucose, 5 g peptone, 5 g yeast extract, 5 g CaCO3 per liter of artificial seawater) were inoculated with strain WMMB272 and shaken (200 RPM, 28 °C) for seven days. Two hundred fifty ml baffled flasks (12 × 50 ml) containing 50 ml ASW-A S2
each were inoculated with 1 ml seed culture and were incubated (200 RPM, 28 °C) for seven days. Two L flasks (20 × 500 ml) containing 500 ml medium ASW-B (5 g soluble starch, 20 g glucose, 10 g soybean flour, 2 g peptone, 2 g yeast extract, 4 g NaCl, 0.5 g K2HPO4, 0.5 g MgSO4*7H2O, 2 g CaCO3 per L of 50% artificial seawater) were inoculated with 5 ml of the 50 ml culture. Strain WMMB272 was cultivated in 10 L ASW-B media followed by extraction with CHCl3 (3 x), which afforded a total of 1.2 g CHCl3 extract. The CHCl3 extract (300 mg) was subjected to a Sephedex LH-20 open column eluted using CHCl3/MeOH (1:1) followed by collection of 13 fractions. The fractions were tested against E. coli and Bacillus subtilis. The active fractions (fr. 3 and fr. 4) were subjected to RP-Phenyl HPLC and yielded bottromycin D (1) 24.3 mg (with minor impurity) at tR 12.6 min. The HPLC conditions were as follows. Conditions: Phenomenex Luna Phenyl-Hexyl 5 µm, 250 mm x 10 mm, Flow rate: 4.5 ml/min, 0.1% acetic acid/H2O (A) and 100% acetonitrile (B), 0~5min: 5~20% B using linear gradient; 5~19min: 20~40% B using linear gradient; 19~20min: 40~100% B using linear gradient; 20~23min: 100% B. For isolation of high purity Bottromycin D (1). using the following improved conditions. Bottromycin D (1) was eluted at tR 18.3 min, 0~5min: 5~15% B using linear gradient; 5~29min: 15~35% B using linear gradient; 29~30min: 35~100% B using linear gradient; 30~34min: 100% B. Isolation of Bottromycin A2. Streptomyces bottropensis was obtained from the USDA and fermented using identical conditions to those previously described.4
Bottromycin A2 was
isolated similarly to bottromycin D (1). A monolithic C18 HPLC column (Phenomenex Onyx monolithic C18 100 x 4.6 mm) was applied instead of the Sephedex LH20 open column due to relatively smaller sample size. The active fractions against E. coli and Bacillus subtilis were subjected to HPLC using the following conditions, which afforded bottromycin A2 at tR 16.0 min. It’s identity was confirmed by HRMS and comparing NMR data with those published.
S3
Conditions: Phenomenex Luna Phenyl-Hexyl 5 µm, 250 mm x 10 mm, Flow rate: 4.5 ml/min, 0.1% acetic acid/H2O (A) and 100% acetonitrile (B), 0~5min: 5~20% B using linear gradient; 5~29min: 20~40% B using linear gradient; 29~30min: 40~100% B using linear gradient; 30~34min: 100% B. LC/MS Profiling of Streptomyces spp. Each strain was inoculated onto ISP2 agar supplemented with artificial seawater in Petri dishes and incubated at 30 °C for 10 days or until sporulation was observed. Two 8 mm cores were removed from the plate, extracted with 2 ml of MeOH for 30 minutes, and the extract was dried using a speedvac. The extract was then dissolved in 100 µL of MeOH and diluted with 1 ml of water prior to solid phase extraction using a 25 mg ABN 1 ml SPE column. The column was washed with 1 ml of water and eluted with 1 ml of MeOH directly into an LCMS vial using a Gilson GX-271 liquid handler.
LCMS analyses were
performed as previously published.5 Data Processing and PCA. Molecular formulas were predicted using Bruker SmartFormula™ algorithm under these parameters: ratio of elements H/C: 0~3; rings plus double bonds: -0.5~40; the nitrogen rule and ions of even electron configuration needed to be fulfilled. PCA was performed using Bruker ProfileAnalysis 2.0 software. Finding molecular features was applied to LC/MS data under these parameters: S/N threshold: 5; correlation coefficient threshold: 0.7; minimum compound length: 10 spectra; smoothing width: 1. MS peak finder used the following parameters: using the same width as used in the acquisition; S/N threshold: 5; relative intensity threshold (base peak): 0.1%; absolute intensity threshold: 100. The bucket generation was performed under the following parameters. The LC/MS data sets were evaluated in a time range from 120 s to 840 s and in a mass range from m/z 200 to 1500. Advanced bucketing was employed using Δ RT = 20 s and Δ m/z = 0.02 Da as parameters. Sum of bucket values was applied for normalization in this study, and Pareto scaling algorithm6 was applied.
S4
16S Amplification and Sequencing. Genomic DNA was extracted using the UltraClean Microbial DNA Isolation kit (Mo Bio Laboratories, Inc.). 16S rDNA genes were amplified using
100-200
ng
genomic
DNA
template
with
the
primers
8-27F
(5’
to
GAGTTTGATCCTGGCTCAG) and 1492R (5’ to 3’ GGTTACCTTGTTACGACTT).
3’ The
following PCR conditions were used: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1.5 min, with a final step of 72 °C for 5 min. The PCR bands were excised from the gel and purified using the QIAquick Gel Extraction kit (QIAGEN). Sequencing reactions were performed by the Biotechnology Center at University of Wisconsin-Madison and reactions were sequenced with an ABI 3730xl DNA Analyzer. The phylogenetic tree was constructed using Tree Builder at the Ribosomal Database Project site (http://rdp.cme.msu.edu/) using the default settings.
Molecular Modeling Calculations. Molecular modeling calculations were performed on a Dell Precision T5500 Linux workstation with a Xeon processor (3.3 GHz, 6-core). Low energy conformers were obtained using Spartan 10 software (MMFF, 10000 conformers examined). The low energy conformer for each compound was analyzed using Gaussian 09 for geometry optimization and NMR calculations (B3LYP/6-31G(d,p)). NMR shifts were referenced to TMS and benzene using the multi-standard (MSTD) approach. Molecules were modeled in the gas phase. Determination of Amino Acid Configurations. L- and DL-FDLA were synthesized as previously reported.7 Bottromycin D (1) (0.3 mg) was hydrolyzed with 6 N HCl (1 ml) for 4 h at 110 °C and dried under vacuum. The acid hydrolysate was dissolved in 100 µL H2O and split into two equal portions. Each portion was mixed with 1 N NaHCO3 (20 µL), acetone (110 µL), and 20 µL of L- or DL-FDLA (10 mg/ml in acetone). Each solution was stirred for 1 h at 40 °C. The reaction was quenched with 1 N HCl (20 µL) and dried under vacuum. A portion of each
S5
product was dissolved in MeOH:H2O (1:1) for LCMS analysis. Separation of the derivatives was achieved with a Phenomenex Kinetex C18 reversed-phase column (2.6 µm, 150 x 2.1 mm) at a flow rate of 0.2 ml/min and with a linear gradient of H2O (containing 0.1% formic acid) and MeOH (90:10 to 3:97 over 29 min). The absolute configuration of the amino acids was determined by comparing the retention times of the L- and DL-FDLA derivatives, which were identified by MS. Minimum inhibitory concentration (MIC) determination. The following strains were used for MIC determinations: ATCC 29213 a Methicillin – Sensitive Staphylococcus aureus (MSSA), and ATCC 33591 a Methicillin – Resistance Staphylococcus aureus (MRSA). A 2-fold dilution was performed in a 96-well plate using bottromycin D (1) [16 µg/ml] in DMSO. The concentrations used for this assay were in a range from 2 – 0.004 µg/ml. The assay was performed in duplicates, each plate containing triplicates of bottromycin D (1) and vancomycin as well. Assays were incubated at 33 ºC for 18 – 20 hours. In order to identify the mode of action of bottromycin D (either bactericidal or bacteriostatic), a sterile swab was used to sample each well that showed inhibitory activity and it was inoculated onto a LB plate overnight at 33 ºC. According with the results, MSSA and MRSA responded equally to bottromycin D (1). Both strains were inhibited at the highest concentration tested (2 µg/ml) in these experiments. The results showed that bottromycin D (1) is a bacteriostatic agent at the concentrations tested in these assays. In order to confirm the mode of action of this compound, increasing the concentration of bottromycin D (1) is required since bactericidal agents can show bacteriostatic activity when they are used in very low concentrations. Bottromycin D (1): white solid; [α]25D -17 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 206 (4.45), 242 (3.62) nm; IR (ATR) υmax 3269, 2970, 2361, 2343, 1738, 1650, 1510, 1415, 1218 cm-1; HRMS [M + H]+ m/z 795.4228, Calcd for C40H59N8O7S, m/z 795.4221. S6
Whole Genome Sequencing and Assembly of the bst Gene Cluster. WMMB272 was grown in 50 ml of TSB (tryptic soy broth, MP Biomedicals) at 30 °C and 200 rpm for 12 days. Cells were harvested by centrifugation and DNA was extracted using published methods.8,9 Precursor peptides were amplified from the genomic DNA using primer pairs bstA1F and bstA1R, and bstA1SF and bstA1SR (see Table S3 for primer list). Purified genomic DNA was prepared as ~330 bp inserts and sequenced at the University of Wisconsin-Madison sequencing core on an Illumina HiSeq 2000 in a 101 bp paired-end run. Two assembly programs were used – Velvet10 and SPAdes.11 The best Velvet assembly was achieved after filtering the raw reads for quality > 30 phred and length = 101 bp with Sickle.12 A k-mer value of 55 was used, with velvetg parameters: -ins_length 330 -ins_length_sd 60 -cov_cutoff 6.3 -exp_cov 1000. The best SPAdes assembly was achieved after filtering the raw reads for quality > 30 phred and length > 40 bp with Sickle. Default parameters were used except that k-mer values of 21, 33, 55, 61 and 71 were employed. Contigs containing genes from the bottromycin gene cluster were identified by TBLASTN searches against both assemblies using the proteins scab_56611−scab_56711 (see Table 1) as queries. Contigs of interest from both assemblies were assembled together in Sequencher (Gene Codes, Ann Arbor, MI) to give four contigs (see Figure S3). The order of these contigs was confirmed by PCR experiments (see Table S3 and Figure S2). Subsequent Sanger sequencing of the PCR products allowed the determination of the full gene cluster sequence (deposited at GenBank). Construction of bst Mutant Strains. The bstA precursor was cloned at the BamH1/ Xba1 site of pSET152 using standard protocols.13
Mutants were constructed using the QuikChange
mutagenesis kit (Stratagene) with primers bstA2 and bstA5. All plasmids were submitted for Sanger sequencing for confirmation. Chemically competent E. coli strain ET12567/pUZ8002 was transformed with pSET152 derivatives and allowed to grow overnight on LB agar plates S7
supplemented with apramycin (50 µg/ml), chloramphenicol (25 µg/ml), and kanamycin (50 µg/ml). Intergeneric conjugation was performed according to the methods of Nybo, et al.9 with some modifications. Spores were collected from plates of WMMB272 (7−9 days old) grown in ISP2 (BD Biosciences) supplemented with 20 g/L NaCl. The plates were flooded with TSB media and the spores were scraped off the plates and filtered through a sterile cotton filter. These were then counted under a microscope using a hemacytometer, washed in TSB, and concentrated. Spores (10 8−109) were used for each conjugation experiment. The WMMB272 spores were then germinated by heat shock treatment at 50 °C for 10 min and incubated while shaking at 30 °C for 3−4 h. At this point, overnight cultures of the E. coli ET12567/pUZ8002 transformed with the desired pSET152 plasmid were harvested and washed by centrifugation in LB (BD Biosciences) 2 to 3 times to get rid of antibiotics. The E. coli cells were then suspended in 100−200 µL of TSB and mixed with the germinated WMMB272 spores. The mixtures were plated in mannitolsoyflour (MS) agar. After 16−20 h at 30 °C, the plates were flooded with 1 ml solution containing nalidixic acid (2.5 µg) and apramycin (1.25 µg) to select for Streptomyces transformants. The plates were then incubated at 30 °C until colonies appeared (10 days or more). Growth of transformants and chemical extraction.
Transformants were picked and
transferred to MS plates with apramycin (25 µg/ml) for further selection. Upon growth, these were then transferred to 5 ml of ASW-B media and incubated at 30 °C at 200 rpm for 3 days. 500 µL of the seed cultures were then transferred to 50 ml ASW-B and grown for 13 days under the same conditions. After 13 days, the culture broths were extracted with 3 equal volumes of chloroform. The chloroform layer was dried under vacuum and the crude extracts were passed through a C18 plug and analyzed by LC/MS, with high-resolution detection by TOF-MS in
S8
positive mode. Accurate mass LC-MS analyses were carried out using a Waters Micromass QTOF Micro integrated LC-MS system employing positive ion ESI mode with an ion source temperature of 100 °C, a desolvation temperature of 450 °C, desolvation with nitrogen gas at a flow rate of 400 L/h, and a cone voltage of 40 V. Chromatography for LC/MS was carried out using a Kinetix C18 column (2.1 × 50 mm; 2.6 µm particle size; Phenomenex, Inc.) at a flow rate of 0.3 ml/min. The mobile phase system consisted of 0.1% formic acid (solvent A) and acetonitrile (solvent B) using the following gradient: 0-1 min, 10% B; 1-17 min, 10-100% B linear gradient; 17-21.5 min, 100% B; 21.5-22 min, 100-10% B linear gradient.
Figure S1. Discovery of New Compounds from Strain WMMB272. The scores plot shows the analysis of 50 bacterial LCMS profiles. Strains that group together tend to have similar secondary metabolite profiles, and strains that separate have unique secondary metabolite profiles as was the case with WMMB-272. The loadings plot is geometrically related to the scores plot; hence the points on the upper right quadrant of the loadings plot represent compounds that cause the statistical variance that makes WMMB-272 unique. Each point is linked to a dynamically linked table containing HRMS measurements. Compound 4 is bottromycin D (1), and we propose that compound 5 is a bottromycin D analog that contains one less CH2 group. Compounds 1, 2, and 3 were previously reported and discovered independently by us and Fenical and co-workers.5,14
S9
Figure S2. Schematic representation of the four contigs obtained from genome sequencing against a map of the bst cluster. Below these are shown the positions of PCR amplicons used to confirm the contig order and the sequences between contigs, along with the primers used (see Table S3).
S10
Table S1. NMR Data for Bottromycin D (1), conformer 1. Amino Acids Gly
cis-3Me-Pro
Ala
no.
1
H NMRa,b
1-N
3.98 br s
2
3.84 m 3.60 br d (J = 11.9 Hz)
3 4-N
13
C NMR
15
Nc
114.2 47.8
Key 1H-13C HMBC
Key ROESY
C3
H-15, H-16, H318~20
C21, C3
H-9, H-29
169.0
5
3.82 m 3.52 m
47.1
C6, C7, C9
6
2.01 m 1.72 m
30.3
C5, C7, C8, C9
7
2.53 m
38.0
C6, C8, C9
8-H3
1.08 d (J = 7.0 Hz)
15.4
C6, C7, C9
9 10 11-N
4.04 d (J = 8.3 Hz)
65.2 174.3
C5, C6, C7, C10
H-11, H-2
C10, C12
H-9
7.64 m
123.2
12
2.88 m
56.4
C10, C13
13-H3
1.52 d (J = 7.5 Hz)
14.9
C12, C14
H-15
t-Leu1
14 15-N
7.25 overlapped
C14
H-1, 13-H3, H3-18~20
4.60 d (J = 10.6 Hz)
54.4 32.9 27.7 157.1
C17, C21, C18~C20
H3-18~20, H-23
C16, C17
H-1, H-15, H-16
t-Leu2
16 17 18~20-H3 21 22-N 23
3.90 s
70.3
C21, C24, C25~C27, C28
H3-25~27, H-16, H-29
1.01/0.97 s
35.4 27.7 172.5
C23, C24
H-23, H-29
C28, C39 C28, C31, C32, C33,
H-2, H3-25~27 H3-32, H-34/38
Me-Phe
Thia-βAla
172.0
1.01/0.97 s
110.8
24 25~27-H3 28 29-N 30
6.99 d (J = 8.23 Hz) 4.99 m overlapped
57.4
31
3.43 m
41.8
C30, C32, C33, C34/C38, C39
32-H3 33 34, 38
1.36 d (J = 7.5 Hz)
C30, C33
7.39 br d (J = 7.6 Hz)
15.3 141.7 128.5
35, 37
7.34 overlap
128.3
C34, C36
36 39 40-N
7.23
127.1 171.8
C34, C35
41 42
113.8
7.08 br m
C35
121.6
C39
5.64/5.60
48.0 169.5
C42
44
7.68/7.64
142.9/142.5
C42
45
7.26/7.25
119.5/119.8
C42
47 48 49-H3
2.90, 3.00 overlap
39.2 170.4/171.1 52.1/52.6
C41, C42, C48
43-N
a
3.72/3.71
C4
in CDCl3. bδ (ppm) 600 MHz. cData were extracted from the 1H-15N HSQC spectrum.
S11
H-34/38
Table S2. NMR Data for Bottromycin D (1), conformer 2. Amino Acids
no.
Gly
1-N 2
cis-3-MePro
5.27 (J = 10.9 Hz) 3.91 m 3.37 m
7 8-H3 9
4.30 d (J = 9.0 Hz)
10 11-N
Nc
111.4 44.5
Key 1H-13C HMBC
Key ROESY
C3 C21, C3
H-23, H3-25~27
45.9
C6, C7, C9
30.6
C5, C7, C8, C9
37.5 15.1
C6, C8, C9 C6, C7, C9
65.2
C5, C6, C7, C10
8.13 d (J = 9.5 Hz)
125.6
C10, C12
H-7, H-13, H-16
12
5.08 m
45.5
C10, C13
H3-18~20, H-16
1.35 d overlapped
18.0 170.9
C12, C14
H-11
15-N
6.42 d (J = 11.2 Hz)
C14
H3-18~20, H-29
16 17
3.93 d (J = 11.2 Hz)
57.1 36.0
C17, C21, C18~C20
H-12, H3-18~20
18~20-H3
1.05 s
26.5
C16, C17
H-15, H-16
C21, C24, C25~C27, C28
H-1, H3-25~27
C23, C24
H-1, H-23, H-29
121.8
158.0
22-N 3.68 s
24
Thia-β-Ala
15
13-H3 14
23
Me-Phe
C NMR
170.0
21 t-Leu2
13
168.2
3.48 m 3.02 m 1.83 m 1.46 m 2.55 m 1.10 d (J = 7.0 Hz)
6
t-Leu1
H NMRa,b
3 4-N 5
Ala
1
68.9 35.5
25~27-H3 28 29-N
0.73 s 6.75 d (J = 6.4 Hz)
C28, C39
H-15
30
4.58 m overlapped
59.1
C28, C31, C32, C33,
H3-32, H-34/38, H-40
31
3.27 m
41.6
32-H3 33
1.33 overlapped
17.0 142.2
C30, C32, C33, C34/C38, C39 C30, C33
34, 38
7.44 br d (J = 7.6 Hz)
129.0
C35
35, 37 36 39
7.35 overlap 7.23
127.5 127.1 170.8
C34, C36 C34, C35
40-N 41
7.08 br m 5.64/5.60
42
26.9 171.4 118.6
121.6 48.0
C39 C42
169.5
43-N
a
44 45
7.68/7.64 7.26/7.25
142.9/142.5 119.5/119.8
C42 C42
47 48
2.90, 3.00 overlap
39.2 170.4/171.1
C41, C42, C48
49-H3
3.72/3.71
52.1/52.6
C4
in CDCl3. bδ (ppm) 600 MHz. cData were extracted from the 1H-15N HSQC spectrum.
S12
Table S3. Primers used in this study. Name
Sequence (5′−3′)
Description
bstAF
GGATCCATGAACAGCGCGACGACG
precursor peptide with 54 nt regulatory region
bstAR
CTAGATCAGACTGACTGGTCGGTGTCG
precursor peptide with 54 nt regulatory region
bstASF
GGATCCATGGGACCAGCGGTCGTATTC
precursor peptide
bstASR
CTAGATCAGACTGACTGGTCGGTGTCG
precursor peptide
bstA2
CTGACATGGGACCAGTAGTCGTATTCGACTG
Site directed mutagenesis primer
bstA3
CAGCGGTCGTATTCACCTGTATGACGGCG
Site directed mutagenesis primer
bstA4
CGGTCGTATTCGCCTGTATGACGGC
Site directed mutagenesis primer
bstA5
CAGCGGTCGTATTCAACTGTATGACGGC
Site directed mutagenesis primer
57F
GTCCGGTAGGCGGATGGTTG
bstC
577F
CACGTTGTTCCACGGCATCG
bstJ
1985R
GACGACTTGGACCACCTGTG
bstH
439R
ACGGGCTACCGGCAGATGTG
bstH
2060F
GCCGGTAGAGGACCAGGTCG
bstH
1817R
CCTTCAACACCTACGCCGTGG
bstF
2654F
CACTCGCTCACCCTCTCGTGC
bstF
866F
TTCACGGCTCCCTCCTCTGC
bstE
595R
GCGTCAACTCCCGCACGAAC
bstE
481R
CCTGGGCAGGGAGATGCTCG
bstC
2366R
CGGTCCGCAGATAGACCAGC
bstK
Figure S3. Amino acid and nucleotide sequences of the precursor peptide. In blue is the 54 nt regulatory region that was included in the bstA* construct.
S13
Figure S4. Extracted ion chromatograms showing A. WMMB-272 transformed with pSETbstA1 produces bottromycin A2. B. WMMB-272 transformed with pSET-bstA1 still produces bottromycin D (1). C. WMMB272 control does not produce bottromycin A2; D. WMMB272 control does produce bottromycin D (1); E and F are WMMB272 transformed with empty vector and still produce bottromycin D (1).
S14
A.
B.
Figure S5. Accurate mass analysis of bottromycin D (1) (A) and bottromycin A2 (B) produced by the transformants.
S15
References (1) Reasoner, D. J.; Geldreich, E. E. Appl Environ Microbiol. 1985, 49, 1-7. (2) Harrison, P. J.; Waters, R. E.; Taylor, F. J. R. J. Phycol. 1980, 16, 28-35. (3) Maldonado, L.A.; Fragoso-Yáñez, D.; Pérez-García, A.; Rosellón-Druker, J.; Quintana, E.T. Antonie Van Leeuwenhoek 2009, 95, 111-20. (4) Kobayashi, Y.; Ichioka, M.; Hirose, T.; Nagai, K.; Matsumoto, A.; Matsui, H.; Hanaki, H.; Masuma, R.; Takahashi, Y.; Omura, S.; Sunazuka, T. Bioorg. Med. Chem. Lett. 2010, 20, 611620. (5) Hou, Y.; Braun, D. R.; Michel, C. R.; Klassen, J. L.; Adnani, N.; Wyche, T. P.; Bugni, T. S. Anal. Chem. 2012, 84, 4277-4283. (6) Van den Berg, R. A.; Hoefsloot, H. C.; Westerhuis, J. A.; Smilde, A. K.; Van der Werf, M. J. BMC Genomics 2006, 7, 142-57. (7) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (8) Keiser, T; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Streptomyces Genetics; The John Innes Foundation: Norwich 2000. (9) Nybo, S. E.; Shepherd, M. D.; Bosserman, M.; Rohr, J. Curr. Protocols Microbiol, 2010, 10E.3.1−10E3.26. (10) Zerbino, D. R.; Birney, E. Genome Res., 2008, 18, 821−829. (11) Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A. A.; Dvorkin, M.; Kulikov, A. S.; Lesin, V. M.; Nikolenko, S. I.; Pham, S.; Prjibelski, A. D.; Pyshkin, A. V.; Sirotkin, A. V.; Vyahhi, N.; Tesler, G.; Alekseyev, M. A.; Pevzner, P. A. J. Comp. Biol., 2012, 19, 455−477. (12) Sickle; Joshi, N. http://github.com/najoshi/sickle. (13) Sambrook, J.; Russell, D. W. Molecular Cloning − A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. (14) Fukuda, T.; Miller, E. D.; Clark, B. R.; Alnauman, A.; Murphy, C. D.; Jensen, P. R.; Fenical, W. J. Nat. Prod. 2011, 74, 1773−1778.
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S6. 1H NMR (600 MHz, CDCl3) of 1
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S7. 1H NMR (500 MHz, DMSO-d6/acetonitrile-d3) of 1
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S8. 13C NMR (125 MHz, CDCl3) of 1
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S9. gCOSY (600 MHz, CDCl3) of 1
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S10. gHSQC (600 MHz, CDCl3) of 1
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S11. gHMBC (600 MHz, CDCl3) of 1
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S12. ROESY (600 MHz, CDCl3) of 1
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S13. 1H-15N HSQC (600 MHz, CDCl3) of 1
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S14. Bottromycin D (1) conformational studies.
Bottromycin D, 2:1 CD3CN:DMSO, 10 °C
Bottromycin D, 2:1 CD3CN:DMSO, 25 °C
Bottromycin D, CDCl3, 25 °C
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S15. Bottromycin D (1) conformational studies.
Bottromycin D, 4:1 CD3CN:DMSO, 10 °C
Bottromycin D, 4:1 CD3CN:DMSO, 25 °C
Bottromycin D, CDCl3, 25 °C
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