For Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517

Supplemental Methods Construction of plasmid pJTU4676SY for site-directed mutagenesis—The construction of pJTU4676SY which was used for introduction of the point mutations into PyrG-KR domain is illustrated in Figure S1. Related plasmids are listed in Table S2. Site directed mutagenesis was accomplished using mutagenic primers (Table S3) and the KOD-PlusMutagenesis Kit (Toyobo). The constructs were verified by DNA sequencing. Construction of ∆pyrU and ∆pyrA Mutants—pyrU and pyrA were inactivated using a standard PCR targeting strategy based on the λRed recombination functions and the aac(3)IV/oriT cassette (1). The gene replacements were confirmed by PCR and the mutated plasmids were introduced into S. pyridomyceticus by intergeneric conjugation using E. coli ET12567/ pUZ8002. Apramycin selection on COM medium produced double crossover mutants HTT5 (∆pyrU) and HTT6 (∆pyrA) (Figure 3, 4), respectively. Complementation of the ∆pyrU and ∆pyrA Mutants—PCR-amplified pyrU was digested using NdeI-BamHI, and pyrA was digested with NdeI-EcoRI. The resulting fragments were cloned downstream of the PermE* promoter into pJTU2170, digested with the corresponding restriction enzymes to yield the complementary plasmids pJTU4652 (for pyrU) and pJTU4637b (for pyrA), respectively (Table S2). They were then introduced into the respective mutant strains by interspecific conjugation. PCR was used to verify the presence of the kanamycin resistance gene and of the insert fragments in the exconjugants. The restored pyridomycin production was assessed by HPLC-MS. Pyridomycin Fermentation, Isolation, HPLC-MS Analysis and Bioassay—A seed culture of S. pyridomyceticus, wild type or recombinant mutants, was grown in a 250 ml flask containing 30 ml of YEME broth and incubated shaking (220 rpm) at 30 °C and for 2 days. The seed cultures were then inoculated at 1% (V/V) into fermentation medium (2.5% glucose, 1.5% soybean meal, 0.5% NaCl, 0.05% KCl, 0.025% MgSO4·7H2O, 0.3% K2HPO4, 0.3% Na2HPO4·12H2O, pH 7.2), and incubated at 30 °C , 220 rpm for 3 days (2). The fermentation broth was extracted twice with equal volumes of ethyl acetate, and the combined extract was concentrated in a vacuum evaporator to produce a solid residue. The residue was dissolved in 1 mL acetonitrile and subjected to LC-ESI-MS (Agilent 1100 series LC/MSD trap system, Agilent Technologies, Santa Clara, CA), using a ZORBAX RX-C18 column (5 μm, 4.6×150 mm, Agilent) with pure pyridomycin as standard for the analysis of pyridomycin production. The column was equilibrated with 80% solvent A (0.1% formic acid in water) and 20% solvent B (0.1% formic acid in acetonitrile), and developed with a 30-min linear gradient from 20% B to 80% B at a flow rate

of 0.5 mL/min and UV detection at 305 nm.The mass spectrometer was run in positive ion detection mode and set to scan between 100 and 800 m/z.
Antibacterial activity of pyridomycin was tested by bioassay using the Oxford cup-plate method (3).The indicator strain was Mycobacterium smegmatis mc 2 155 grown in LBG medium (LB medium containing 0.5% glycerol and 0.05% Tween 80) at 37 °C.For the bioassays, Oxford cup-plates were placed on LBG plates containing 60 μL of a 18 h culture of M. smegmatis.S. pyridomyceticus wild type or recombinant mutants were similarly cultured, extracted and then appropriate amounts of the respective extracts were filled into the Oxford cup-plates.The pyridomycin standard was used as a control.After incubation at 37 °C for approximately 16 hr, an inhibition zone around the cup-plate indicated antibacterial activity.Precursor-directed biosynthesis of pyridomycin analogues-Seed cultures in YEME medium were inoculated with S. pyridomyceticus spores and inoculated at 30 °C, 220rpm for 2 days.Then the fermentation medium was inoculated with 1% seed culture and incubated at 30 °C.After 24 hr, pH neutral solutions of 3HPA or its analogous aromatic acids was added into the culture to final concentration of 2 mM.After incubation for another 48 hr, the culture supernatant was harvested, extracted twice with equal volumes of ethyl acetate, and the resulting organic extracts were combined, evaporated to dryness and finally dissolved in acetonitrile.LC-QTOF-MS analysis was performed on an Agilent 1200 series LC/MSD trap system in tandem with a 6530 Accurate-Mass Q-TOF MASS Spectrometer with an ESI source.Large scale preparation, purification of the pyridomycin standard-To prepare a pyridomycin standard, 3L fermentation broth was extracted three times with 1.5L ethyl acetate.The organic supernatants were combined and concentrated on a vacuum evaporator.The raw sample was purified by silica gel column chromatography (chloroform-methanol, 20:1).Each fraction was tested for alkaloid compounds using bismuth potassium iodide, and the collected fractions containing pyridomycin were concentrated, dissolved in methanol, and loaded onto a preparative silica gel TLC plate.The resulting TLC plates were developed in chloroform-methanol (10:1), and the pyridomycin spot was determined by UV scanning at 350 nm and bismuth potassium iodide.The purity of the final product was verified by analytical HPLC.Q-Tof MS confirmed the product identity (observed 541.2303 and calculated 541.2293 for C 27 H 33 N 4 O 8 + ).
Overproduction and Purification of Recombinant Proteins-The plasmids pJTU4655, pJTU4655(S47A), and pJTU4637 were separately transformed into E. coli BL21 (DE3) and the resulting E. coli cells were grown to OD 600 of 0.5~0.6 at 37 °C in LB medium supplemented with 50 μg/mL kanamycin.After adding 0.4 mM IPTG, the cultures were incubated for an additional 4 h at 30 °C.Cells were harvested by centrifugation, resuspended in 20 mL buffer A (150 mM NaCl, 20 mM Tris•HCl pH8.0), and lysed by sonication.The debris was removed by centrifugation twice for 30 min at 15000×g.After filtration through a 0.45 μm filter (ANPEL Scientific Instrument Co., Shanghai), the clarified supernatant was loaded on a 1-mL HisTrap HP column that was pre-equilibrated with buffer A. Further purification of the recombinant proteins was carried out using an Amershan Biosciences FPLC system following a gradient from 0 to 100% buffer B (20 mM Tris•HCl, 500 mM Imidazole, 150 mM NaCl, pH 8.0) for 20 column volumes at a flow rate of 1 mL/min.The fractions containing the desired protein were combined, concentrated, and the buffer was changed to buffer C (50 mM Tris，0.5 mM EDTA，50 mM NaCl，5% glycerol, pH 7.9) using a Millipore 3 K MWCO Ultra filter for PyrU and PyrU(S47A).The purified proteins were aliquoted and stored at -80 °C.
PyrA was further purified using a 1-mL HiTrap DEAE FF column connected to an Amershan Biosciences FPLC and a gradient from 0 to 1 M NaCl in 20 mM Tris•HCl buffer (pH 8.0) for 20 column volumes at the flow rate of 1 mL/min.The fractions containing pure PyrA were combined, dialyzed against buffer C, aliquoted, and frozen at -80 °C.
The purity of the isolated proteins was examined by SDS-PAGE (15% for PyrU and PyrU(S47A) and 10% for PyrA).Protein concentration was determined by the Bradford assay (4) (Tiangen, Beijing).Table S1  pristinaespiralis and its homologs that were used for degenerate primers design.Top: multiple amino acid sequence alignments of 3-hydroxypicolinic acid:AMP ligase.SnbA and VisB are responsible for pristinamycin and virginiamycin biosynthesis, respectively; the conserved regions used to design for degenerate primers are labeled.Bottom: the PCR products from contig 2.

Figure S3. A 20 kb deletion of contig 2
Figure S3.A 20 kb deletion of contig 2 proves its involvement in pyridomycin biosynthesis.A. Construction of mutant HTT7 generated by double crossover gene replacement.B. HPLC profile of a, pyridomycin standard, and b-d, extracts from the mutant HTT5 and wild-type strain, respectively.The slanted arrows indicate the pyridomycin peak which is missing in b and c.

Figure S4 .
Figure S4.Sequence alignment of the KR domain.Red dots, Lys139, Ser163 and Tyr176 from the catalytic triad.