Materials and reagents
All reagents, kits, and chemicals used for molecular biology and chemistry experiments were obtained from commercial sources, including Sigma-Aldrich Shanghai Trading Co Ltd. (Shanghai, China), Thermo Fisher Scientific (Waltham, MA, USA), New England Biolabs, and used without further purification unless otherwise specified. Enzymes were purchased from Takara Biotechnology (Dalian, China) or Vazyme Biotech (Nanjing, China) unless otherwise specified.
All plasmids and strains used in this study are listed in Supplementary Table 1. The composition of culture media used in this study is listed in Supplementary Table 8. Streptomyces lividans 66 and S. monomycin NRRL B-24309 were obtained from the Agricultural Research Service Culture Collection (Peoria, IL, USA). E. coli BW25141 and WM6026 were provided by Dr. William Metcalf (University of Illinois, Urbana, IL, USA). S. cerevisiae HZ848 and the plasmid pRS416 were provided by Dr. Huimin Zhao (University of Illinois, Urbana, IL, USA). The plasmid pET-15b was provided by Dr. Huan Wang (Nanjing University, Nanjing, China). All promoters, RBS, primers, and oligonucleotides were synthesized by Tsingke biological technology (Fuzhou, China).
Instrumentation
1H NMR spectra were measured on a JEOL-ECS-400 (400 MHz) spectrometer. Chemical shifts are reported in ppm from the solvent resonance or tetramethylsilane (TMS) as the internal standard. Splitting patterns were recorded as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad peak. 13C NMR spectra were recorded on a JEOL-ECS-400 (100 MHz) spectrometer with complete proton decoupling. Chemical shifts were reported in ppm from the solvent resonance as the internal standard. 31P NMR spectra were recorded on a JEOL-ECS-400 (162 MHz) spectrometer. Chemical shifts were recorded in ppm from 85% H3PO4 (0 ppm) resonance as the external standard. The spectra were analyzed using the Delta NMR software or MestRenova software version 8.1.1. The LC/MS system used was a Waters® Xevo® G2-XS Qtof high-resolution mass spectrometer coupled with an ACQUITY® UPLC® I-Class Bio System. Data were collected in positive or negative ion mode using a SONAR quadrupole window, which provides a full scan MS over the TOF mass range of 100–1000 Da. The data was processed post-acquisition in UNIFI. HPLC measurements were performed on a 7900HT Essentia Pre LC-16P (Essentia, Kyoto, Japan) equipped with a UV detector (SPD-16). PCR was performed on a Bio-Rad T100TM Thermal Cycler using Phusion® or Q5® High-Fidelity DNA polymerase. Real-time PCR was performed using an Applied Biosystems 7900HT Fast Real-Time PCR System. High-speed refrigerated centrifuge Avanti was performed using an American Beckman Coulter Co., Ltd J-26S XP. The pH measurements were performed on a PHS-3B digital pH meter (Shanghai Hongyi Instrument Co. Ltd.).
Whole-genome sequencing and analysis
Genomic DNA of the strain S. monomycin NRRL 24309 was extracted with the SDS method65. The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit® 2.0 Fluorometer (Thermo Scientific). A total amount of 1 μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, the DNA sample was fragmented by sonication to a size of 350 bp. DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing with further PCR amplification.
At last, PCR products were purified (AMPure XP system), and libraries were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified using real-time PCR. The whole genome was sequenced using Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. Illumina PCR adapter reads, and low-quality reads from the paired-end were filtered by the step of quality control using our own compiling pipeline. All good quality paired reads were assembled using the SOAP denovo66, SPAdes67, and ABySS68 into a number of scaffolds. Then the filter reads were handled by the next step of the gap-closing. The assembled genomic data for each strain in fastq format were uploaded to Rapid Annotation Using Subsystem Technology (RAST) server for annotation28. The annotated genomes were analyzed using RAST tools and antiSMASH29.
Streptomycete cultivation and expression analysis
Spores from S. lividans 66 transformed with different combinations of alp genes and promoters were inoculated into MYG medium (Supplementary Table 8) at 28 °C with constant shaking (200 r.p.m.). After 72-h cultivation, the total RNA was isolated with a TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cDNA was performed using M-MLV First-strand cDNA Synthesis Kit (Invitrogen, USA). Real-time PCR was performed with SYBR Green PCR Master Mix (Bio-Rad). Primers were designed using the online tool provided by Integrated DNA Technologies (https://www.idtdna.com/scitools/Applications/RealTimePCR/). The reaction system was generated by gently mixing 10 µL of 2 × SYBR Green Mix, 1 µL of cDNA templates, 1 µL of each primer at a concentration of 10 pmol µL−1, and 7 µL of ddH2O in each well of the 96-well plate. Amplification was carried out using the following program: 3 min at 50 °C, 3 min at 95 °C for one cycle; 20 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C for 30 cycles with a final cycle of 10 min. The endogenous gene hrdB, encoding RNA polymerase sigma factor, was employed as the internal control for all real-time RT-qPCR experiments. The transcriptional levels of all target genes were normalized against the expression level of the reference gene.
DNA manipulation and gene cluster reconstruction
Gene cluster fragments (each 2000–3000 bp) were PCR-amplified from the genomic DNA isolated from S. monomycin NRRL B-24309 using an UltraClean Microbial DNA Isolation Kit (Qiagen). All primer sequences were summarized and listed in Supplementary Data 1. The S. cerevisiae helper fragment was PCR-amplified from the plasmid pRS416, whereas the E. coli helper fragment and the S. lividans helper fragment were PCR-amplified from the plasmid pAE4. Following electrophoresis, all PCR products were individually gel-purified from 0.7% agarose using Qiagen Gel Purification Kit. Each gene expression cassette, including gene, promoter, insulator, or RBS, was assembled by overlap extension PCR (OE-PCR). To ensure yeast homologous recombination’s high efficiency, the cassette was designed to generate a ~200 bp overlap region with adjacent fragments. Individual cassettes (2–3 kb, 300 ng each) were gently mixed and precipitated with 100% ethanol. After air drying, the resulting DNA pellet was resuspended in 4 µL of deionized water and stored at −20 °C for yeast transformation.
Yeast transformation
The concentrated mixture of DNA fragments was electroporated into the yeast S. cerevisiae HZ848. An aliquot of 0.5 mL of S. cerevisiae HZ848 cells was inoculated into a 50 mL YPAD liquid medium (Supplementary Table 8), and the culture was shaken at 250 rpm and 30 °C for about 5 h until the OD600 value reached 1.0. Yeast cells were collected by high-speed centrifugation (5000 × g) at 4 °C for 5 min. The supernatant was immediately removed, and the cell pellet was washed with 50 mL ice-cold deionized water, followed by washing with ice-cold 1 M sorbitol and finally resuspended in 250 mL ice-cold sorbitol. An aliquot of 50 µL of yeast cells together with a 4 µL DNA mixture was electroporated in a 0.2-cm chilled electroporation cuvette at 1.5 kV with a time constant of 5.0–5.2 ms, followed by immediate addition of 1 mL pre-warmed (30 °C) YPAD medium to respend the transformed cells. The cells were shaken at 30 °C and 250 rpm for 1 h. The transformed cells were then collected by high-speed centrifugation, washed with 1 M sorbitol several times to remove the YPAD medium, and finally resuspended in 1 ml sorbitol at room temperature. Aliquots of 50–100 µL were spread on SC-Ura plates, and the plates were incubated at 30 °C for 2–4 days until colonies appeared69.
Restriction digestion analysis
Several colonies were randomly picked to SC-Ura liquid media and grown at 30 °C and 250 rpm for one day, after which the plasmids were extracted from yeast cells using the TIANprep Yeast Plasmid DNA Kit (TIANGEN, Beijing, China). An aliquot of 50 µL of E. coli strain BW25141 together with a 2 µL isolated plasmid was electroporated in a 0.2-cm chilled electroporation cuvette at 2.5 kV with a time constant of 5.0–5.2 ms, followed by immediate addition of 1 mL pre-warmed (37 °C) SOC medium to respend the transformed cells. The cells were shaken at 37 °C and 250 rpm for 1 h. The cells were spun down, and 800 mL of SOC medium was removed. The cell pellet was resuspended with the remaining 200 mL of SOC medium, and the cells were spread on LB plates supplemented with 50 µg mL−1 apramycin (Apr). The plates were incubated at 37 °C for about 18 h until colonies appeared. Colonies were inoculated into 5 mL of LB media supplemented with 50 µg mL−1 apramycin and grew at 37 °C for 12–16 h. E. coli plasmids isolated from the liquid culture were then verified through at least two separate restriction digests for each plasmid. Usually, appropriate restrictive enzymes were chosen to cut the target plasmid at multiple sites that will result in multiple fragments with obviously various sizes. The reaction mixtures were loaded to 1% agarose gels to check for the correct restriction digestion pattern by DNA electrophoresis.
Conjugation and heterologous expression in S. lividans
The verified plasmids were electroporated to an auxotrophic strain E. coli WM6026 and selected on LB agar plates supplemented with 2, 6-diaminopimelic acid (DAP) and apramycin. These transformants were then used as the donors for conjugative transfer of the assembled plasmids to S. lividans 6670. The verified plasmid was mixed with E. coli WM6026 cells, and the mixture was put into a chilled electroporation cuvette. The cells were electroporated at 2.5 kV and quickly resuspended by adding SOC medium and DAP. Then the transformed cells were cultured in a shaker at 37 °C, 250 rpm for 1 h. The culture was spread on an LB-Apr+-DAP plate, and the plates were incubated at 37 °C for 16 h until colonies appear. A single colony was inoculated from each plate into 2 mL of LB supplemented with 50 µg mL−1 apramycin and 10 mL of 38 mg mL−1 DAP and grew at 37 °C until OD600 reaches 0.6–0.8. The cell culture was centrifugated in an Eppendorf tube, and the cell pellet was washed with the fresh LB medium twice. The cell pellets were resuspended with the fresh LB. The resuspended cells were mixed with S. lividans spores by pipetting and spot 2 mL aliquots onto R2 no-sucrose plates. After all the spotted drops were absorbed entirely into the R2 no-sucrose plates, the plates were incubated at 30 °C for about 18 h. The plates were flooded with a mixture of nalidixic acid and apramycin at a 1 mg/mL concentration. The plates were incubated at 30 °C for about 3–5 days until exconjugants appeared. A single exconjugant was picked and restreaked on ISP2 plates supplemented with 50 µg mL−1 apramycin and grew for about 3–5 days. A single colony was inoculated into the ATCC172 liquid medium supplemented with 50 µg/mL apramycin in a tube for 3 days and then inoculated into 100 mL ISP2 liquid medium supplemented with 50 µg/mL apramycin. The cells were incubated for seven days at 30 °C and 250 rpm.
Expression and purification of AlpH, I, J, G, K, and L
Linear pET-15b was prepared by cutting the circular plasmid with NdeI and PCR-amplifying the resulting product. The PCR reaction (50 μL) contained 1X Fail-Safe buffer G, 50 μM of each primer, 20–40 ng of linear plasmid, and 0.05 U µL−1 Q5 High-Fidelity DNA polymerase. The line plasmid was amplified through the following program on a thermocycler: 98 °C 3 min, 30 cycles of 98 °C 10 s, 58 °C 1 min, 72 °C 5 min, then 72 °C 8 min. The PCR product was digested with the restriction enzyme DpnI and purified with the Wizard® SV kit (Promega).
pET-15b-AlpG (or H, I, J, K, and L) was prepared by amplifying the gene from S. monomycin NRRL B-24309 genomic DNA. The 50 μL reaction contained 1X Fail-Safe buffer G, 1 μM of each primer (Supplementary Data 1), 250 ng gDNA, and 0.05 U µL−1 Q5 High-Fidelity DNA polymerase. The system was run through the following program on a thermocycler: 98 °C 2 min, 25 cycles of 98 °C 15 s, 58 °C 15 s, 72 °C 1 min, then 72 °C 8 min.
The PCR samples were run on a 1% agarose gel and purified using the Wizard® SV kit (Promega). The products (200 ng) were used directly for Gibson assembly with 100 ng linear pET-15b DNA. The 20 μL ligation reactions contained a 1X NEB Gibson master mix. The ligation reactions were incubated at 50 °C for 1 h.
An aliquot (~2 μL) was used to transform chemically competent E. coli DH5α cells. The transformed cells recovered in SOC media for one hour at 37 °C before being plated on LB plates supplemented with ampicillin and grown overnight at 37 °C. Colonies were selected, and the cells were grown overnight at 37 °C. The plasmid was extracted, and the sequence was confirmed by sequencing at Boshang Biotech Company (Fuzhou, China).
The pET-15b expression vectors were chemically transformed into E. coli BL21 Rosetta (DE3) cells. The transformed cells were grown in LB media supplemented with ampicillin (100 μg mL−1) and chloramphenicol (25 μg mL−1) at 37 °C until the OD600 value reached 0.6. The temperature was then adjusted to 18 °C, and the expression was induced by the addition of 0.3 mM IPTG. After 14–20 h, the transformed cells were collected by centrifugation (12,000 × g, 10 min, 4 °C). Cells were flash-frozen in liquid nitrogen and stored at −80 °C. Cells harvested from 1000 mL of LB were resuspended in 25 mL of buffer C (50 mM HEPES pH 7.5, 200 mM KCl, 10% (v/v) glycerol) supplemented with 20 mM imidazole, 0.4 U mL−1 DNase, and 1 mg mL−1 lysozyme at room temperature. Cells were lysed by sonication (3 × 45 s with 10 min rocking periods at 4 °C, and the insoluble debris was removed via centrifugation (35,000 × g, 1 h, 4 °C).
Clarified lysates were affinity purified using a 5 mL nickel-nitrilotriacetic acid (Ni-NTA) column previously equilibrated with buffer C supplemented with 20 mM imidazole. Protein was eluted with buffer C supplemented with 250 mM imidazole. Elution fractions were combined and concentrated using a 30 kDa MWCO centrifugal filter and then buffer exchanged into buffer C with either a NAP-25 column or dialysis tubing following the manufacturer’s instructions. Protein was flash-frozen in liquid N2 and stored at −80 °C for future use.
AlpH and AlpI coupling enzymatic reaction
The reaction mixture (500 µL) contained 1.5 mM thiamine diphosphate, 2 mM Mg2+, 10 µM AlpH, 10 µM AlpI, and 2 mM PEP in 50 mM HEPES buffer (pH 7.5). The reaction was initiated by the addition of 10 µM AlpI. The reaction mixture was incubated at 28 °C for 5 h, subjected to IMAC and HILIC chromatography, and then analyzed by 31P NMR spectroscopy.
AlpJ enzymatic assay
The biochemical reaction was performed in 50 mM HEPES pH 7.25 with 20 µM AlpJ, 2 mM PnAA, 5 mM NADH, and 5% glycerol in a total volume of 500 µL. The reaction mixture was incubated at 28 °C for 16 h, subjected to IMAC and HILIC chromatography, and then analyzed by 31P NMR spectroscopy.
AlpG enzymatic assay
The reaction mixture (500 µL) contained 10 µM AlpG, and 2 mM 2-HEP in 50 mM HEPES buffer (pH 7.5). The reaction mixture was incubated at 28 °C for 2 h, subjected to IMAC and HILIC chromatography, and then analyzed by 31P NMR spectroscopy.
AlpK and AlpL coupling enzymatic assay
The reaction mixture (500 µL) contained 2 mM Mg2+, 1 mM NAD+, 1 mM NADP+, 5 mM l-glutamate or l-aspartate, 2 mM PLP, 10 µM AlpK, 10 µM AlpL, and 2 mM HMP in 50 mM HEPES buffer (pH 7.5). The reaction was initiated by the addition of 10 µM AlpK. The reaction mixture was incubated at 28 °C for 1 h, subjected to IMAC and HILIC chromatography, and then analyzed by 31P NMR spectroscopy.
Phylogenetic analysis
A multiple sequence alignment of six discrete MPnS-related proteins was generated using the Clustal Omega71 web tool (http://www.ebi.ac.uk/Tools/msa/clustalo/) with the default parameters. All phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis (MEGA) X72. Maximum likelihood phylogenetic trees were created in MEGA using the standard parameters. A WebLogo frequency plot was generated from a Clustal Omega alignment of all of the sequences using the standard parameters73.
NMR analysis
The liquid culture was centrifuged, and the pellets were removed immediately. The liquids were evaporated to dryness using a rotary evaporator. The crude sample was prepared by dissolving 1.0–3.0 mg in 250 µL of D2O (Sigma-Aldrich, 99.96% atom%D). NMR spectra were recorded on a JNMR ECZ-400s 400 MHz spectrometer equipped with a 5 mm FG/TH auto-tune probe. Samples were run at 25 °C during acquisition. Standard pulse sequences were set up for each experiment, including 1H, 13C, 31P, and 1H-31P HMBC. Spectra were recorded with the Delta NMR software and analyzed with the software MestReNova 8.1.1.
LC-MS analysis
The crude sample was redissolved in H2O and then sonicated at 0 °C for 5 min. The sample was injected into the system for UPLC–HRMS detection (Waters) with a HILIC column (2.1 × 50 mm 1.7 μm). The gradient was: 0–5 min, 100% B; 5–15 min, 100% B–40% B, 15–20 min, 40% B–100% B; 20–35 min 100% B at a flow rate of 0.2 Ml min−1. Solvent A: 10 mM NH4HCO3; Solvent B: 10 mM NH4HCO3, 90% Acetonitrile (ACN). Samples were analyzed by precursor ion screening in negative mode (monitoring m/z 50).
Isolation and purification of argolaphos A (3) from S. monomycini B-24309
Streptomyces m. B-24309 was cultivated in 250 mL of ATCC172 seed medium for 3 days at 30 °C on a platform shaker rotating at 200 rpm before inoculation of 20 L of solid ISP4 plates. After incubating at 30 °C for 10 days, the agar-solidified medium was liquefied by repeated freezing and subsequent thawing. Argolaphos were isolated and purified using the modified version of the previous procedure15. Briefly, The resulting supernatant (15 L) was generated by filtration on a Büchner funnel before extensive concentration via rotary evaporation. A total volume of 2 l of 90% methanol was added and shaken vigorously by hand. The supernatant was filtered and concentrated to dryness and the residue dissolved in 50 mL of 0.1% aqueous acetic acid, which was applied to a 40 mL Fe(III) IMAC column. The phosphonates were eluted from the column with 100 mM aqueous NH4HCO3. Several rounds of 31P NMR guided size-exclusion chromatography were used to isolate and purify the phosphonates. The elute chromatographed over different glass Sephadex LH-20 columns (from 40 mm × 1400 mm to 10 mm × 1800 mm gel bed) eluted with distilled H2O to produce many fractions. Based on 31P NMR analysis of each fraction, fractions containing phosphonates were combined and dried with a rotary evaporator to give the sample, which was dissolved in 0.5 mL of distilled H2O and further purified using a 10 mm × 250 mm HILIC column (Atlantis®HILIC Silica, 5 µm) and gradient elution, yielding compounds 3 (3 mg) and 4 (1 mg).
Reduction of argolaphos A (3) to compound 8
Indium-mediated reduction of 3 to the corresponding amine 8 was accomplished using the previous method26. Briefly, compound 3 (0.5 µmol) was put into a 2:1 solution of ethanol and saturated aqueous. NH4Cl (1 mL) in a 5 mL round bottom flask equipped with a Claisen condenser and a magnetic stirring bar. An appropriate amount of In powder (2 equiv.) was added, and the system was heated to reflux for about 8 h. After the reaction monitored by HILIC HPLC was complete, the mixture was cooled down, filtered over Celite, concentrated, and dissolved in distilled H2O. Furthermore, the product was purified using a 10 mm × 250 mm HILIC column (Altantis® HILIC Silica, 5 µm) and a gradient elution, yielding the compound 8 (95% yield). The gradient used was: 20 min at 100% solvent B (0.1 formic acids in ACN) then a linear gradient to 60% solvent A (0.1% formic acids in distilled H2O) over 40 min. The flow rate was 3 mL min−1. The retention time was 23 min.
Marfey’s analysis
A dried sample of ~0.1 mg of the peptide was dissolved in 2 mL of 6 N HCl in an ampule and heated to reflux for 14 h in an oil bath pot74. The hydrolysate was dried entirely under N2 in a glass vial. 3.6 μmol of a 1% acetone solution of FDAA (N-(5-fluoro-2,4-dinitrophenyl)-d-alaninamide) and 20 μmol of 1 M NaHCO3 were added to the peptide to initiate the derivatization reaction in the vial. The reaction mixture was then heated with an HH-S digital thermostat water bath (Jiangsu Jintan Medical Instrument Factory) at 40 °C for 50 min under agitation and cooled to room temperature. Then, the reaction was quenched by adding 20 μmol of 1 M HCl and then 1 mL of MeOH. The mixture without isolation was analyzed with a Waters® Xevo® G2-XS Qtof high-resolution mass spectrometer coupled with an ACQUITY® UPLC® I-Class BioSystem using a reverse-phase C18 column, and retention times were compared with those of the corresponding amino acid standards. The eluent A comprises 0.05% HCOOH in ACN and eluent B as the water was used through a linear gradient elution mode (eluent A, 10–60%, 60 min) at a flow rate of 0.4 mL min−1.
Absolute configuration was determined by comparing the retention time of derivatives from commercially available amino acids and spiked experiments with standard amino acids. Therefore, the absolute configuration of valine and N5-hydroxyl arginine in compound 3 was determined to be L.
Quantification of AMP (2) in the culture
Quantification of 2 in this study was based on the previous methods with minor modifications75,76. Briefly, samples (1 mL) for 2 measurement were taken after 5-day bacterial culture and was centrifuged (10,000 rpm, 5 min, 4 °C). The pellets were discarded, and the supernatant was then stored at −80 °C for further use. Extraction of 2 was carried out by adding 1 mL of CH3OH: CH3CN: H2O (2:3:1) to 0.1 mL of the samples. The samples were subjected to sonication for 5 min and high-speed centrifugation at 12,000 rpm for 8 min at 4 °C. The pellets were discarded, and the supernatant was treated in a high vacuum centrifuge. The dried samples were dissolved in 50 µL of distilled water and filtered for UPLC/MS analysis. The standard procedure was applied for all the above samples. Dense samples with a higher concentration than the maximum limit of quantitation have to be diluted with the appropriate amount of distilled water. Five repeat injections were used as technical replicates to validate the UPLC/MS method used in this experiment. Three replicates were used to detect the robustness of the AMP extraction method used. From each culture, three samples were extracted, and each sample was analyzed twice.
For the 2 analysis using UPLC/MS, 10 µL of the sample was injected into an ACQUITY BEH amide 1.7 µm column (2.1 × 50 mm; Waters, USA). The autosampler was run at 12 °C, and the column oven was kept at 28 °C. The following solvents were designed for the LC analysis. Solvent A: 66% H2O, 33% CH3CN, 10 mM CH3COONH4, 0.04% NH4OH, pH 9; solvent B: 10% H2O, 90% CH3CN, 10 mM CH3COONH4, 0.04% NH4OH, pH 9. The LC program was carried out at 0.4 mL min−1, and the injection volume was kept at 10 μL. The program started from 0% B for 2 min, followed by a gradient from 0% B to 100% B within 10 min. Then 100% B was kept for 2 min, followed by a constant balance at 0% B for 5 min. The identification and detection of 2 were achieved by the Agilent UPLC-ESI-QTOF-MS system in positive mode. HRMS acquiring parameters were set below: capillary voltage (3 kV); drying gas temperature (300 °C); drying gas flow rate (12 L min−1); nebulizer pressure (35 psi). The scan source parameters were set below: skimmer voltage (65 V), fragmentor voltage (175 V), and octupole RF peak voltage (700 V). Tandem MS acquisition of daughter molecular ion for 2 was treated with fixed collision energy 30 eV in the positive mode. All standard curves were produced using linear regression for the 2 quantifications. The linearity was calculated using nine concentrations, 2, 4, 8, 16, 32, 64, 128, 256, 512 ng mL−1 (n = 5). The signal-to-noise (SN) ratio required for the limit of detection (LOD) and limit of quantification (LOQ) was built using the SN script conducted in Analyst 1.6.2 software. A time window of 0.5 min prior to the peak of AMP was chosen as noise, and the peak of AMP itself was defined as the signal in a time window of 0.1 min. Both LOD and LOQ were calculated by the lowest concentration of the spiked sample with an SN ratio of at least 3 and 10, respectively. The accuracy and precision were estimated at different concentration levels in each matrix. A recovery of 70–120% is acceptable in this study.
Chemical synthesis of 1 via a biomolecular nucleophilic substitution (SN2) reaction
AMP (2) (1 mmol) and haloacetic acids (1 mmol) were added to 0.5 M NaHCO3 solution and stirred at 50 °C (or 90 °C) for 3 h.
Chemical synthesis of 1 by reductive amination
To AMP (2) (1 mmol) and glyoxylic acid (1 mmol) in MeOH-AcOH (10:1, 5 mL) was added α-picoline-borane (1 mmol), and the mixture was run for 1 h under agitation at room temperature62. The reaction was monitored by 31P NMR.
Purification of phosphonates
In our study, the isolation and purification of phosphonates include four basic steps: 70–100% MeOH precipitation, IMAC chromatography, Sephadex LH-20, and HILIC HPLC with minor modifications according to the property and the concentration of target compounds. Here we provide a brief description of the whole procedure.
After the culture or the reaction was completed, distilled H2O was immediately added, and the solution was partitioned with EtOAc twice. The organic layer was discarded. The aqueous fraction was concentrated by rotary evaporation, and an appropriate amount of methanol (MeOH) was added to reach a final concentration of 70–100% MeOH. The pellet was removed by high-speed centrifugation, and the supernatant was dried using rotary evaporation. The step can be repeated several times to remove the highly polar components as much as possible.
The supernatant above was dried using a vacuum centrifuge, and the pellet was dissolved in a 0.1% acetic acid solution and subjected to a Fe(III) IMAC gravity column. The IMAC-packed column was equilibrated entirely with 0.1% aqueous acetic acid. The column was then washed successively with 5 volumes of 0.1% aqueous acetic acid after loading the sample onto the column. The phosphonate materials were easily eluted from the iron IMAC column with 200 mM NH4HCO3. The eluate from the column was utterly concentrated with a rotary evaporator.
The pellet was dissolved in distilled H2O and was then put onto a Sephadex LH-20 gravity column (40 × 1400 mm gel bed) eluted with distilled H2O (flow: 200 mL h−1; fractions: 10 mL) to generate many fractions. Based on the 31P NMR analysis of each fraction, fractions with the same or similar 31P NMR profile were combined. All fractions were concentrated with a rotary evaporator. The step can be repeated several times by changing the different sizes of the Sephadex LH-20 column.
The fraction containing the target compound was dissolved in distilled H2O and further purified using a 10 × 250 mm HILIC column (Atlantis®HILIC Silica, 5 µm) and a gradient elution, yielding pure 1. The gradient used was: 20 min at 100% solvent B (0.1% TFA in ACN) then a linear gradient to 60% solvent A (0.1% TFA in distilled H2O) over 40 min. The flow rate was 3 mL min−1.
Statistical analysis and data visualization
All statistical tests were computed in the Sigmaplot 12.5, and data visualization was performed using the package ggplot2 in R software v.3.1.3.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
