Different Biosynthetic Pathways to Fosfomycin in Pseudomonas ...

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species Seung Young Kim,a Kou-San Ju,a William W. Metcalf,a Bradley S. Evans,a Tomohisa Kuzuyama,b and Wilfred A. van der Donka,c Institute of Genomic Biology, University of Illinois at Urbana—Champaign, Urbana, Illinois, USAa; Biotechnology Research Center, University of Tokyo, Tokyo, Japanb; and Howard Hughes Medical Institute and Department of Chemistry, University of Illinois at Urbana—Champaign, Urbana, Illinois, USAc

Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

F

osfomycin is an FDA-approved antibiotic containing epoxide and phosphonate functional groups (Fig. 1) (8). The compound blocks peptidoglycan biosynthesis by inhibiting UDPGlcNAc enolpyruvyl transferase (MurA) (29, 38). In the clinic, fosfomycin is used predominantly against acute cystitis and sometimes gastrointestinal infections (1, 2, 15, 43, 45, 56). Previous studies in several laboratories have supported the fosfomycin biosynthetic pathway shown in Fig. 1 in Streptomyces fradiae and Streptomyces wedmorensis (37, 50, 59, 60). Like in nearly all phosphonate biosynthetic pathways (39), the first committed step in fosfomycin biosynthesis in these streptomycetes is the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) (22, 23, 33). This thermodynamically unfavorable transformation is made feasible by linking it to the irreversible decarboxylation of PnPy to phosphonoacetaldehyde (PnAA) by a thiamine-dependent decarboxylase (41). Subsequent reduction to 2-hydroxyethylphosphonate (2-HEP) (51, 59), methylation to generate 2-hydroxypropylphosphonate (2-HPP) (20, 34, 59, 60), and epoxide formation (19, 25, 37, 49, 62) complete the biosynthesis of fosfomycin (Fig. 1). All enzymatic steps in this pathway have been reconstituted in vitro, except for the methyl transfer step, which is putatively catalyzed by the radical S-adenosylmethionine (SAM) methyltransferase Fom3 (58, 59, 61). Attempts to achieve in vitro activity with purified Fom3 enzymes from streptomycetes have thus far been unsuccessful. Given the highly unusual chemistry of the reaction that Fom3 is proposed to catalyze, experimental verification of its proposed role would be valuable. Fosfomycin and various analogs are also produced by several pseudomonads, such as Pseudomonas viridiflava PK-5, Pseudomonas fluorescens PK-52, and Pseudomonas syringae PB-5123 (30, 53). In an attempt to investigate an ortholog of the Fom3 enzymes from these Gram-negative bacteria, the fosfomycin biosynthetic gene cluster from Pseudomonas syringae PB-5123 was identified. Surprisingly, its genome does not contain a gene encoding a Fom3 ortholog, nor does it contain a gene encoding a PnPy decarboxyl-

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ase. We show that the first and last steps of fosfomycin biosynthesis in P. syringae are the same as those reported for previously investigated pathways in streptomycetes but that an entirely different set of enzymes is used to convert PnPy to 2-HPP. MATERIALS AND METHODS Materials. Chemical reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Pittsburgh, PA) and were used without further purification. Medium components were purchased from Thermo Fisher Scientific or VWR (West Chester, PA). Bacterial strains, plasmids, and the sequences of PCR primers are listed in Table 1. BP clonase was obtained from Invitrogen (Carlsbad, CA). Escherichia coli strains were grown in Luria-Bertani (LB) broth (3) at 37°C. Tryptone-yeast extract medium (TY; 1% tryptone, 0.5% yeast extract) and minimal-salts broth (MSB) (55) containing 1% (vol/vol) Balch’s vitamins (17) were used for growth of Pseudomonas strains. For plates, MSB was solidified with 1.8% (wt/vol) Noble agar, and LB and TY were solidified with 1.6% (wt/vol) Bacto agar. Antibiotics were added at the following concentrations for plasmid selection and maintenance; kanamycin (Kan), 50 ␮g ml⫺1; apramycin (Apr), 25 ␮g ml⫺1; chloramphenicol (Cm), 15 ␮g ml⫺1; and gentamicin, 5 ␮g ml⫺1. DNA isolation and manipulation. All cloning was performed by established methods (46). Endonucleases and T4 DNA ligase were purchased from Invitrogen (Carlsbad, CA) and New England BioLabs (Ipswich, MA). Shrimp alkaline phosphatase was purchased from Roche

Received 2 January 2012 Returned for modification 30 January 2012 Accepted 9 May 2012 Published ahead of print 21 May 2012 Address correspondence to Wilfred A. van der Donk, [email protected]. This paper is dedicated to the memory of Professor Haruo Seto, honoring his pioneering contributions to the natural-product field. Supplemental material for this article may be found at http://aac.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.06478-11

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FIG 1 Biosynthetic pathway toward fosfomycin in Streptomyces. Steps that are confirmed by experimental data with purified enzymes are shown with solid arrows, whereas putative transformations are indicated with dashed arrows. MeCbl, methylcobalamin.

Diagnostics GmbH (Mannheim, Germany). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Plasmids and fosmids were isolated using Qiagen (Valencia, CA) miniprep or maxiprep kits. High-molecular-weight chromosomal DNA of P. syringae for library construction was prepared using standard protocols (21). PCR amplifications were performed with FailSafe PCR PreMix buffers (Epicentre, Madison, WI). PCR screenings were performed with KOD Hot Start polymerase (Novagen, EMD Chemicals Inc., Gibbstown, NJ). Plasmids used for functionalization of Pseudomonas aeruginosa were purified with a Fermentas GeneJet miniprep kit (Glen Burnie, MD), and DNA fragments were purified with a Fermentas GeneJet gel extraction kit. An UltraClean microbial DNA isolation kit (Mo Bio, Carlsbad, CA) was used for the purification of genomic DNA for genome sequencing. DNA sequencing was carried out at the Roy J. Carver Biotechnology Center at the University of Illinois, Urbana—Champaign, on an ABI 3730XL capillary sequencer. E. coli S17-1 ␭-pir was used to introduce plasmids into Pseudomonas strains by conjugative matings. Strains of S17-1 ␭-pir containing plasmids for mobilization were cross-streaked with Pseudomonas strains and incubated at 30°C for 16 h. Cells were then resuspended in 10 ml of MSB, homogenized by vortexing, and plated onto MSB plates containing 10 mM sodium succinate and the appropriate antibiotics. Exconjugants were purified by repeated single-colony isolations. Genome sequencing of P. syringae PB-5123. The genomic DNA of P. syringae PB-5123 was sequenced on a Roche 454 GS-FLX system at the Roy J. Carver Biotechnology Center at the University of Illinois, Urbana— Champaign. The sequence reads were assembled using the Newbler program (454 Life Sciences). Construction of a library of genomic DNA of P. syringae PB-5123. Genomic DNA was prepared from P. syringae PB-5123 grown in 100 ml of LB for 18 h at 30°C in a 0.5-liter baffled flask. The cells were harvested and homogenized, and genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen, Valencia, CA). The genomic DNA was analyzed by field inversion gel electrophoresis (FIGE) and determined to be primarily DNA fragments of ⬎200 kb. The prepared genomic DNA was partially digested with Sau3AI by treating the DNA with serial dilutions of the enzyme. The fraction with the highest yield of ⬃35- to 50-kb fragments was determined by FIGE. The fosmid vector pJK050 was prepared by sequential NheI digestion, treatment with shrimp alkaline phosphatase, and BamHI digestion. The genomic DNA fraction was ligated with digested pJK050 overnight at 16°C, followed by ethanol precipitation and packaging into lambda phage using a MaxPlax packaging extract according to the manufacturer’s instructions (Epicentre, Madison, WI). E. coli WM4489 cells were transfected with the packaged library and plated on LB–12-␮g/ml Cm agar plates. WM4489 is an E. coli DH10B derivative engineered to provide copy-number control for pJK050 fosmids through regulation of the trfA33 gene (encoding plasmid replication protein) by a rhamnoseinducible promoter (14). Individual colonies (⬃1,300) were picked into

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standard 96-well plates, with each well containing a single 3-mm glass bead (to aid mixing) and 200 ␮l of LB–12 ␮g/ml Cm. The samples were grown overnight with shaking at 37°C. Library screening. Cultures (10 ␮l) from 48 clones were pooled, boiled in water (100 ␮l), and used as the template for PCR mixtures containing 500 nmol of primers FomScreenF and FomScreenR (Table 1) and KOD Hot Start DNA polymerase in 1⫻ PCR premix G, with melting at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s for 30 cycles in total. Pools that yielded DNA bands corresponding to the psf1 gene fragment were then individually screened by PCR to identify individual clones containing the putative fosfomycin gene cluster. Fosmid DNA was isolated from single positive clones grown overnight in 5 ml LB–12 ␮g/ml Cm– 0.2% rhamnose using a Qiagen miniprep kit. The purified fosmids were then individually recombined in vitro with similarly purified pAE4 using a BP clonase kit according to the manufacturer’s instructions (14). The reaction mixtures were used to transform E. coli WM4489, and successfully recombined plasmids were identified by the appearance of colonies on LB plates containing Cm and Apr. Clones were screened by PCR for the presence of several genes in the putative cluster (psf1, psf2, psf3, psf5, psf6, and psf7) using the primers listed in Table 1. One fosmid (46-2-11) containing a 22.5-kb insert was positive for the presence of the psf1-3 and psf5-7 genes and was sequenced. E. coli transformants containing 46-2-11 were used as donors for conjugal transfer of the fosmid to P. aeruginosa PAO1-LAC after retrofitting and strain construction (see below). Modularized system for expanding ␾C31 integration. A system was created to extend the range of target strains amenable for ␾C31 integration by modification of a mini-Tn5 transposon system to include a native ␾C31 attB site. Plasposon pTnMod-RCm (12) was first tailored to confer gentamicin instead of chloramphenicol resistance. The 841-bp DNA fragment containing the gentamicin acetyltransferase gene (aacC1) from SacI-digested pTnMod-OGm was purified and ligated to the ⬃3.5-kb fragment of similarly digested pTnMod-RCm to produce pKSJ248. The orientation of aacC1 was verified by restriction digestion with BsrGI and SpeI. A DNA fragment containing the ␾C31 attB site was amplified from Streptomyces coelicolor A3(2) by PCR using primers PhiC31attBF-2 (with restriction sites XbaI, SpeI, and NotI; Table 1) and PhiC31attBR (with restriction sites HindIII, PacI, and MfeI; Table 1). The 291-bp product was purified, digested with XbaI and HindIII, and ligated with similarly digested pUC18 to produce pKSJ256. After verification of the desired construct by sequencing, the ␾C31 attB site was excised from pKSJ256 using SpeI and PacI, and the resulting fragment was ligated with similarly digested pKSJ248 to produce pKSJ259. Heterologous expression of the fosfomycin biosynthetic gene cluster. P. aeruginosa PAO1-LAC was functionalized with the ␾C31 attB site by introducing pKSJ259 through conjugative transfer from E. coli S17-1 ␭-pir. A gentamicin-resistant derivative containing the ␾C31 attB site (verified by PCR) was designated KSJ554. Fosmids 46-2-11 and pJK50AE4 were integrated into KSJ554 by conjugative transfer from E. coli S17-1 ␭-pir. Single gentamicin-, chloramphenicol-, and apramycin-resistant colonies were selected and designated strains KSJ570 (harboring the fosmid without the insert) and KSJ629 (harboring fosmid 46-2-11). The presence of the intact fosfomycin gene cluster in KSJ629 was verified by diagnostic PCRs using the primers in Table 1. Heterologous production of fosfomycin detected by GC-MS. Volatile, silylated derivatives of fosfomycin were prepared by the addition of 200 ␮l N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)–1% trimethylchlorosilane (TMCS) (Aldrich) (54) to a 2-ml amber glass sample vial and then addition of 100 ␮l sample (either fosfomycin standard, crude spent medium of P. syringae PB-5123, crude spent medium of P. aeruginosa KSJ570, or crude spent medium of P. aeruginosa KSJ629) dissolved in methanol. Derivatization was performed for 40 min at 70°C. Reaction mixtures were then analyzed on an Agilent 6890N gas chromatographmass spectrometer (GC-MS) at the Roy J. Carver Metabolomics Center at the University of Illinois Urbana—Champaign. Sample introduction was


Two Biosynthetic Pathways to Fosfomycin

TABLE 1 Bacterial strains, plasmids, and sequences of oligonucleotide primers for PCR experiments used in this studya Strain, plasmid, or primer name

Relevant characteristics or sequence

Reference or source

E. coli DH5␣ ␭-pir BL21(DE3) S17-1 ␭-pir WM4489

Cloning host; contains ␭-pir thi Expression host Host for plasmid mobilization; contains ␭-pir thi DH10B derivative

57 11 14

P. syringae PB-5123

Fosfomycin producer

53

P. aeruginosa PAO1-LAC KSJ554 KSJ570 KSJ629

PAO1 derivative; lacIq⫹ delta(lacZ)M15⫹ tetA⫹ tetR⫹ Derivative of PAO1-LAC, contains ␾C31 attB Gmr Derivative of KSJ554, pJK50-AE4 integrated in PhiC31 attB Gmr Cmr Aprr; contains 46-2-11 Derivative of KSJ554, fosmid 46-2-11 integrated in ␾C31, attB Gmr Cmr Aprr

ATCC 47085 This study This study This study

Plasmid for retrofitting pJK050 derivatives for conjugal transfer and integration; ␭ attP, ␾C31 int, ␾C31 attP, Aprr Plasmid for constructing fosmid libraries; ␭ attB Clmr Cloning vector, Ampr Expression vector, Kanr Plasposon, pMB1 ori Gmr Plasposon, R6K ori Cmr Plasposon (pTnMod-RGm), R6K ori Gmr pUC18 containing ␾C31 attB Ampr pKSJ248 containing ␾C31 attB Gmr Derivative of pJK050, recombined with pAE4, contains fosfomycin gene cluster from PB-5123; Clmr Aprr Derivative of pJK050, recombined with pAE4; Aprr Clmr pET26b containing psf1, Kanr pET26b containing psf2, Kanr

14

Plasmids pAE4 pJK050 pUC18 pET26b pTnMod-OGm pTnMod-RCm pKSJ248 pKSJ256 pKSJ259 46-2-11 pJK050-AE4 pPSF1-his pPSF2-his Primers FomScreenF FomScreenR Psf1FP Psf1RP Psf2FP Psf2RP Psf3F Psf3R Psf5F Psf5F Psf6F Psf6R Psf7F Psf7R PhiC31attBF-2 PhiC31attBR

14 Invitrogen 12 12 This study This study This study This study This study This study This study

5=- GGGATGTCATCAATAACGCATGCAG-3= 5=-GGGTTACCGTGCATACAAACGCTCAGC-3= 5=- GGGCATATGTCATCAATAACGCATGCAG-3= 5=-GGGAAGCTTTTACCGTGCATACAAACGCTCAGC-3= 5=- GGGCATATGGAAGGCGTCATGAATATTCGC-3= 5=-GGGGAATTCTCATTTCAGCGTCGCATGCAG-3= 5=-GGGCATATGCGGGCGCAACGAAGTGAGCAG-3= 5=-GGGAAGCTTTCATGACGCCTTCCATGCTTTG-3= 5=-GGGCATATGCCAGACACTTTGATCGCCAGC-3= 5=-GGGAAGCTTTCAAGCGTAATCGACTGCCAG-3= 5=-GGGGAATTCCGAGAATGCTCGCTTCTTCATG-3= 5=-GGGGGATCCTTACTTGTGCAGCTCTTCAGCG-3= 5=-GGGGGATCCCCCGTTCACGCCAGGAGTCG-3= 5=-GGGGCGGCCGCTCACTCGCTCAAGAACACGGTTCC-3= 5=-CGTTCTAGACTAGTGCGGCCGCTCACCGTGACCACCGCGCCCAGCGG-3=b 5=-CGTAAGCTTAATTAAcaattgGGTGATGGTGCCGCCGCCACCGTTG-3=c

a

Ampr, ampicillin resistant; Aprr, apramycin resistant; Cmr, chloramphenicol resistant; Kanr, kanamycin resistant. Restriction sites are underlined. Italics, bold, and underlining indicate the restriction sites for XbaI, SpeI, and NotI, respectively. c Italics, bold, and underlining indicate the restriction sites for HindIII, PacI, MfeI, respectively. b

via split injection onto an HP-5 (5% phenyl methylpolysiloxane) column (30 m, 0.25-mm inner diameter, 0.25-␮m film thickness). The injector temperature was 250°C. The initial column temperature was 40°C, and the temperature was held at this temperature for 5 min after injection before it was increased to 230°C at 15°C min⫺1. The temperature was held at 230°C for the remainder of the 27-min program. Under these conditions, derivatized fosfomycin was detected at 14 min. Peaks at this retention time were analyzed for fosfomycin by the presence of an ion with m/z 210 with a diagnostic fragmentation pattern. NMR spectroscopy. All nuclear magnetic resonance (NMR) experiments were performed at the Varian Oxford Center for Excellence in


NMR spectroscopy at the University of Illinois, Urbana—Champaign. The presence of phosphonates was determined using 1H-decoupled 31P NMR spectroscopy. All spectra were collected in H2O supplemented with 20% D2O as a lock solvent. The 31P NMR spectra were externally referenced to an 85% phosphoric acid standard (0 ppm). Spectra were acquired at room temperature on a Varian Unity Inova-600 spectrometer equipped with a 5-mm Varian 600DB AutoX probe with ProTune accessory for detection at the 31P frequency. Preparation of recombinant Psf1 and Psf2. The psf1 and psf2 genes were amplified by PCR with the forward primer Psf1FP (Table 1; the NdeI site is underlined) and the reverse primer Psf1RP (Table 1; the HindIII site

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is underlined) and with the forward primer Psf2FP (Table 1; the NdeI site is underlined) and the reverse primer Psf2RP (Table 1; the BamHI site is underlined), respectively. The PCR products were then cloned into a pET26b vector to produce pPSF1-his and pPSF2-his, respectively (Table 1). The expression plasmids containing psf1 and psf2 were used to transform E. coli BL21(DE3) cells. These strains were then grown in LB medium with Kan to an optical density at 600 nm of 0.8, and protein expression was induced by the addition of isopropyl-␤-D-thiogalactoside (IPTG) to 0.1 mM. Cultures shaken at 20°C overnight were harvested by centrifugation and stored at ⫺80°C. For purification, cell pellets were thawed and resuspended in 45 ml of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 20 mM imidazole, pH 7.5). Lysozyme was added to a concentration of 1 mg/ml, and the resulting suspension was incubated on ice for 30 min. Cells were disrupted by two passes through a French press (20,000 lb/in2), and debris was removed by centrifugation. The resulting supernatant was slowly agitated with 5 ml (bed volume of resin) of Ni-nitrilotriacetic acid resin (Qiagen, Valencia, CA) prewashed with lysis buffer at 4°C for 3 h. The suspension was loaded onto a column, and the flowthrough fraction was collected. The resin was washed with lysis buffer containing 20 mM imidazole until the concentration of proteins in the eluent decreased substantially, as judged by a visual test with Bradford reagent. The bound protein was eluted with a buffer containing 250 mM imidazole. The desired fractions, as detected by SDS-PAGE, were pooled and concentrated using an Amicon Ultra YM-10 centrifugal filter unit (Millipore, Billerica, MA). The protein sample was loaded onto a PD-10 desalting column (GE Healthcare, Piscataway, NJ) and eluted with 50 mM Tris-HCl, 200 mM NaCl, pH 7.5, as per the column manufacturer’s instructions. Phosphonomethylmalate formation by Psf2. The phosphonomethylmalate synthase activity of the His6-tagged Psf2 was assayed by 31P NMR spectroscopy and Fourier transform mass spectrometry (FTMS). The assay mixture (1 ml) contained 50 mM HEPES-K⫹ (pH 7.2), 5 mM MgCl2, 0.3 mM acetyl coenzyme A (Ac-CoA), 5 mM PEP, 10 ␮M Psf1, and 50 ␮M Psf2. The reaction mixture was incubated at 30°C for 1 h. Then, the proteins were removed using a Microcon YM-10 filter unit, and the solution was lyophilized. Samples were reconstituted in 90% acetonitrile containing 10 mM ammonium bicarbonate and analyzed on a Thermo Fisher 11T linear trap quadrupole-FTMS with an attached Agilent 1200 high-pressure liquid chromatography system. Reaction products (100 ␮l) were separated on a Zic pHILIC column (2.1 by 150 mm; SeQuant, Umeå, Sweden) using 90% acetonitrile containing 10 mM ammonium bicarbonate (solvent B) and 10 mM ammonium bicarbonate (solvent A). The mobile phase was held at initial conditions (100% B) for 2 min, followed by a linear gradient down to 40% B over 15 min, a 3-min linear gradient back to starting conditions, and a final 15-min reequilibration. FTMS data were acquired in negative mode, scanning m/z 100 to 700 at a resolution of 100,000 (full width at half maximum). Data were analyzed manually using the Qualbrowser application of Xcalibur software (Thermo Fisher Scientific, San Jose, CA). Nucleotide sequence accession number. The sequence of the insert in fosmid 46-2-11 has been deposited in GenBank under accession number JX102649.

RESULTS

Fosfomycin gene cluster in P. syringae PB-5123. To identify the genes encoding fosfomycin biosynthesis, genomic DNA was prepared from the fosfomycin producer P. syringae PB-5123 (53) and the genome was sequenced using 454 sequencing. Assembly of the sequence reads (estimated coverage, 95-fold) yielded 124 contigs totaling 5,828,174 bp. A gene (psf1; Fig. 2) that encodes a protein with high homology to PEP mutase (PepM), the enzyme that generates the POC bond in the first step of nearly all phosphonate natural-product biosynthetic pathways by converting PEP to PnPy (Fig. 1) (39), was identified. The genome of P. syringae PB5123 contains only one gene (psf1) homologous to pepM. Sur-

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FIG 2 (Top) Fosfomycin biosynthetic gene cluster in S. fradiae; (Bottom) DNA fragment of the genome from P. syringae PB-5123 that includes the biosynthetic gene cluster for fosfomycin production. Red, the biosynthetic genes that are present in S. fradiae, S. wedmorensis, and P. syringae; blue, the previously characterized resistance gene fomA and its ortholog psf7 in P. syringae; green, a gene encoding a citrate synthase-like protein similar to enzymes involved in the biosynthesis of FR-900098 and phosphinothricin. For the other genes in the bottom panel, see Table 2.

rounding the psf1 gene are several open reading frames (ORFs) with homology to genes present in the phosphonate biosynthetic pathways of FR-900098 (14) and phosphinothricin (5, 48) (Fig. 2 and Table 2). As expected from previous studies on the epoxidase responsible for the last step of fosfomycin biosynthesis in both streptomycetes (37) and pseudomonads (36, 40) (Fig. 1), a gene encoding the epoxidase was present (here designated psf4). An ortholog of the fomA gene (psf7), which has previously been shown to encode a protein that confers self-resistance by phosphorylating fosfomycin (16, 31, 35, 42), was also present. However, a gene encoding a FomB ortholog, another protein involved in self-resistance in fosfomycin-producing streptomycetes by converting the compound to its bisphosphorylated analog (31, 35), was not found in the PB-5123 genome. More surprisingly, genes homologous to fom2 or fom3 that encode enzymes that convert PnPy to 2-HPP in Streptomyces (Fig. 1) were also absent. Instead, one of the ORFs (psf2) encodes a protein that has homology with the citrate synthase-like proteins FrbC and Pms (see Fig. S1 in the supplemental material) that are encoded in the biosynthetic gene clusters of the phosphonate natural products FR900098 and phosphinothricin, respectively (5, 14, 24, 48). These enzymes have been shown to add an acetyl group from Ac-CoA to the carbonyl group of phosphonopyruvate and phosphinopyruvate, respectively (14, 24). Thus, it appears that the unfavorable equilibrium between PEP and PnPy (Keq ⬍ 0.002) (7) is not driven forward by decarboxylation in PB-5123, as in S. fradiae and S. wedmorensis, but instead is driven forward by addition of an acetyl group to the carbonyl moiety of PnPy, similar to the pathways of FR-900098 (14) and phosphinothricin (24, 52). In vitro activity of Psf1 and Psf2. To confirm that the psf1 and psf2 genes encode enzymes with the predicted activities, both proteins were heterologously expressed in E. coli as N-terminally His6-tagged proteins and purified by immobilized metal affinity chromatography (IMAC). Incubation of PEP with both proteins indeed produced phosphonomethylmalate (Pmm), as confirmed by 31P NMR spectroscopy and comparison with an authentic standard prepared using the homologous enzymes from the FR900098 pathway (Fig. 3) (14). Additional peaks in the phosphate ester region of the spectrum (⬃5 ppm to ⫺3 ppm) are from inorganic phosphate, PEP, and impurities present in commercial PEP. When either Psf1 or Psf2 was omitted from reaction mixtures, PEP was not converted to new products, consistent with the observation that the equilibrium between PEP and PnPy favors PEP by more than 500-fold (7).



TABLE 2 Summary of open reading frames in the fosfomycin biosynthetic gene cluster of P. syringae PB-5123a Amino acid identity/ similarity (%)

ORF

No. of amino acids

Protein homologb

psf6 psf9 psf10 psf11 psf1 psf12 psf13 psf4 psf7 psf8 psf3 psf2 psf5 psf14

302 403 339 246 291 197 258 190 261 275 295 380 191 121

Pseudomonas putida F1 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (YP_001268782) Chromobacterium violaceum ATCC 12472 MFS family transporter (NP_900838) Unknown protein Pseudovibrio sp. strain JE062 hypothetical protein (ZP_05084540) Beggiatoa sp. strain PS phosphoenolpyruvate phosphomutase (ZP_01998559) Planctomyces maris DSM 8797 hypothetical protein (ZP_01854986) Burkholderia oklahomensis C6786 hypothetical protein (ZP_02365922) Pseudomonas syringae epoxidase (BAA94418) Pseudomonas syringae phosphotransferase (CAA83855) Pseudomonas syringae unknown (CAA83856) Pectobacterium carotovorum subsp. carotovorum PC1 6-phosphogluconate dehydrogenase (YP_003016070) Burkholderia oklahomensis EO147 trans-homoaconitate synthase (ZP_02358889) Photorhabdus asymbiotica subsp. asymbiotica ATCC 43949 non-heme Fe-binding protein (YP_003042097) Pseudomonas putida GB-1 hypothetical protein (YP_001668139)

a b

96/98 24/44 32/55 52/69 39/53 26/39 100/100 100/100 100/100 30/50 59/76 40/59 95/97

Homologous proteins and their putative functions are listed along with the sequence identity/similarity. The designations in parentheses are the GenBank accession numbers.

Heterologous production of fosfomycin in P. aeruginosa. We next set out to demonstrate that the 12-kb genomic DNA fragment shown in Fig. 2 indeed encodes a functional fosfomycin gene cluster via heterologous expression in P. aeruginosa. We first constructed a large-insert fosmid library from PB-5123 genomic DNA. The fosmids were individually recombined in vitro with pAE4 using BP clonase (14). The fosmid library was used to transform E. coli WM4489, and successfully recombined plasmids were selected on LB agar plates supplemented with Cm and Apr. The library was then screened by PCR with primers specific to psf1. Nine positive clones were obtained, and these were further screened by PCR for the presence of psf2, psf3, psf5, psf6, and psf7 using the primers in Table 1. One fosmid (46-2-11) contained an insert of 22.5 kb with all six genes. Sequencing and annotation of the insert revealed the 12-kb DNA fragment shown in Fig. 2. This clone was then chromosomally integrated into the ␾C31 attB site

of P. aeruginosa strain KSJ554 to test for heterologous production of fosfomycin by this gene cluster. P. aeruginosa strains KSJ570 (negative control; pJK050-AE4 integrated), KSJ629 (46-2-11 integrated), and P. syringae PB5123 (positive control) were analyzed for fosfomycin production by 31P NMR spectroscopy, GC-MS, and liquid chromatography (LC)-FTMS. As reported previously, the native producer generates only very small amounts of fosfomycin (53) (see Fig. 5B and F). P. aeruginosa containing the fosmid with the putative fosfomycin gene cluster also produced small amounts of fosfomycin, detected by 31P NMR spectroscopy, GC-MS, and LC-FTMS analyses (Fig. 4 and 5 and Fig. S2 in the supplemental material, respectively). The negative control did not show any fosfomycin. Thus, the genes shown in Fig. 2 indeed confer the ability to produce fosfomycin. To further corroborate this conclusion, deletion of psf1 from PB-5123 abolished fosfomycin

FIG 3 NMR and FTMS detection of the product of incubation of PEP and Ac-CoA with Psf1 and Psf2. (A) 31P NMR spectrum of 2-phosphomethylmalate (Pmm) formation from Ac-CoA and PEP in the presence of Psf1 and Psf2. (B) LC FTMS analysis for compounds having a mass of Pmm ⫾ 1 ppm. A summed mass spectrum is shown on the right.

FIG 4 NMR detection of fosfomycin produced by P. aeruginosa KSJ629. 31P NMR spectra of partially purified spent medium of P. aeruginosa KSJ629 (A) and the same sample supplemented with fosfomycin authentic standard (B). The resulting spectrum in panel B showed an increase in the relative peak area at 11.9 ppm. The identity of the other phosphonate products is not known. Production of multiple phosphonates upon heterologous expression of phosphonate biosynthetic gene clusters is a common observation (6, 14) and may be a consequence of either buildup of biosynthetic intermediates or detoxification by the heterologous host.


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FIG 5 GC-MS analysis of derivatized fosfomycin. (A to D) Extracted ion chromatograms monitoring for ions with m/z 210 from concentrated extracts of the heterologous producer P. aeruginosa KSJ629 (Me, methyl; TMSO, trimethylsilyloxy) (A), concentrated extracts of the native producer P. syringae PB-5123 (B), authentic fosfomycin (C), and concentrated extracts of P. aeruginosa KSJ570 (negative control) (D). The arrows in panels A to D indicate the presence of an ion with m/z of 210. In panel D, a peak with an m/z of 210 is observed, but it elutes slightly earlier (14.0 versus 14.1 min) and the mass spectrum and fragmentation pattern do not correspond to those of the standard. (E) Mass spectrum of the GC peak at 14.1 min produced by P. aeruginosa KSJ629; (F) mass spectrum of the GC peak at 14.1 min produced by P. syringae PB-5123; (G) mass spectrum of authentic fosfomycin eluting at 14.1 min; (H) mass spectrum of compounds eluting at 14.0 min produced by P. aeruginosa KSJ570. All samples were silylated as described in Materials and Methods.

production (see the discussion and Fig. S2 in the supplemental material). Attempted in vitro reconstitution of Psf3, Psf5, Psf6, and Psf7. Having established that the gene cluster is responsible for fosfomycin biosynthesis, we attempted to determine the biosynthetic pathway used in P. syringae. As discussed above, the first two steps are likely conversion of PEP to Pmm (Fig. 3). Aside from the previously demonstrated epoxidase activity of Psf4 (36, 40), which converts 2-HPP to fosfomycin in the last step of the pathway, the sequences of the proteins encoded by the additional ORFs in the gene cluster do not provide immediate clues for the manner in which Pmm is converted to 2-HPP (Table 2). Psf3 shows sequence homology with an NAD-dependent 6-phosphogluconate dehydrogenase, whereas Psf5 has sequence homology with nonheme iron-dependent proteins (Table 2). We expressed these two proteins as well as another putative dehydrogenase (Psf6) in E. coli as N-terminally His6-tagged proteins, purified all three proteins by IMAC, and evaluated their activity on the last known intermediates in the pathway, Pmm in the forward direction and 2-HPP in the reverse direction. Unfortunately, no activity was detected. The reason may be that these proteins act on other intermediates in the pathway, require specialized cofactors, or act on intermediates that are conjugated to nucleotides. The last possibility is supported by the presence of psf7, which encodes a protein with homology to phosphotransferases. It is possible that Psf7 generates a nucleotide or phosphorylated analog of either fosfomycin itself or one of its biosynthetic intermediates. Indeed, a nucleotide analog of fosfomycin, fosfadecin, has been isolated from Pseudomonas viridiflava PK-5 (30), supporting the potential conjugation. Conjugation of nucleotides to pathway intermediates is also found in

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the biosynthesis of FR-900098 and phosphinothricin. In both cases, a nucleotidyltransferase transfers a CMP moiety from CTP to one of the phosphonate oxygen atoms of a biosynthetic intermediate about halfway into the overall pathway (4, 27). We heterologously expressed Psf7 in E. coli and purified the protein but to date have not been able to detect any activity using a series of nucleotides and potential phosphonate substrates (fosfomycin, PnPy, Pmm, 2-oxopropionylphosphonate). Sequence analyses indicate that Psf10, Psf11, Psf12, Psf13, and Psf15 do not have clear homology with proteins of known function. These gene products may carry out steps in the biosynthetic pathway between Pmm and 2-HPP and generate intermediates that are the actual substrates for Psf3/Psf5/Psf6/Psf7. DISCUSSION

The original goal of this study was the identification of an ortholog in P. syringae of the methyltransferase Fom3 involved in fosfomycin biosynthesis in several streptomycetes. Surprisingly, no gene encoding such an ortholog was found in a draft genome of P. syringae PB-5123, despite the observation that the strain produced fosfomycin, albeit in very small quantities, as had also been observed previously (53). Furthermore, a fosmid containing the open reading frames shown in Fig. 2 (bottom) was able to confer heterologous production of fosfomycin upon P. aeruginosa PAO1-LAC. Collectively, these results establish two distinct biosynthetic pathways to fosfomycin in S. wedmorensis/S. fradiae and P. syringae. As demonstrated by the activity of the purified enzymes Psf1 and Fom4 in this and previous studies (36, 40), the pathways have the first and last steps in common. Sequence alignments show that Fom1 and Fom4 from S. fradiae have 32% and



FIG 6 Proposed biosynthetic pathway toward fosfomycin in P. syringae PB-5123. Steps that are confirmed by experimental data with purified enzymes are shown with solid arrows, whereas putative transformations are indicated with dashed arrows.

35% identity to Psf1 and Psf4, respectively (see Fig. S3 in the supplemental material). These numbers demonstrate that these proteins are only distantly related and that these two pathways are the result of convergent evolution. Whereas the first and last steps are the same, the genes for the decarboxylase Fom2 and methyltransferase Fom3, used by the fosfomycin-producing streptomycetes that have been investigated thus far, are absent in P. syringae. Instead, a longer pathway appears to be used by P. syringae to convert PnPy to 2-HPP. The details of the pathway are still unclear but likely involve the addition of an acetyl group to the carbonyl of PnPy to generate Pmm. A chemically feasible path to fosfomycin from Pmm can be proposed (Fig. 6), taking into account the sequence homology of some of the genes found in the cluster. Hydroxylation of Pmm (possibly by Psf5) would provide intermediate 1. Subsequent decarboxylation of the C-3 carboxyl group with concomitant elimination of the hydroxyl group at C-1, possibly assisted by prior phosphorylation by Psf7, would provide intermediate 2. This reaction sequence would be analogous to phosphorylation prior to decarboxylation by mevalonate 5-diphosphate decarboxylase (26) or sulfation prior to decarboxylation in curacin A biosynthesis (18). Alternatively, concomitant decarboxylation and dehydration without phosphorylation could take place as observed for prephenate dehydratase (9, 47), but the latter reaction has a stronger driving force as a consequence of aromatization of the substrate. None of the proteins encoded by the ORFs of unknown function in P. syringae have sequences that are homologous with known decarboxylases. However, chemically favorable decarboxylation reactions such as those involving substrates with a ␤-carbonyl functionality or with a good ␤-leaving group are catalyzed by a wide variety of enzymes. Examples are the above-mentioned decarboxylation during curacin biosynthesis catalyzed by a thioesterase (18) and the decarboxylation of carboxyphosphonopyruvate to phosphinopyruvate by carboxy-PEP mutase (44). After the


decarboxylation/elimination reactions, compound 2 would tautomerize to compound 3, and this ␤-keto acid could undergo another decarboxylation to produce 2-oxopropionyl phosphonate (2-OPP) promoted by one of the ORFs of unknown function. Subsequent reduction, possibly by Psf3, would generate 2-HPP, which is the substrate for Psf4-catalyzed formation of fosfomycin (36, 40). As discussed above, some of the putative intermediates in Fig. 6 may be conjugated to phosphate or nucleotide groups. Regardless of whether the proposed biosynthetic route in Fig. 6 is correct, the pathways in both Streptomyces strains as well as in Pseudomonas add additional carbon atoms to ultimately arrive at 2-HPP. For the pathway in streptomycetes, the homologation step is performed by a radical SAM-dependent methyltransferase (Fom3), whereas a citrate synthase-type reaction is used in pseudomonads (Psf2). Of note, the two pathways utilize the two previously identified strategies of driving the unfavorable equilibrium of PEP and PnPy forward (39), decarboxylation and addition of Ac-CoA. These two distinct pathways to fosfomycin are an interesting and unusual example of convergent evolution in natural-product biosynthesis. Similar evolutionarily distinct pathways toward the same chemical structures have been described in primary metabolism (e.g., menaquinone, thiamine, lysine, and isopentenyl diphosphate) (10, 13, 28, 32), and a few cases have been described in secondary metabolism (e.g., polyketides) (10). ACKNOWLEDGMENTS We thank Shionogi Co. Ltd. for providing P. syringae PB-5123, Jonathan Dennis (University of Alberta) for providing plasposons pTnModR-Cm and pTnMod-OGm, and Alfred Pühler (University of Bielefeld) for providing plasmid pK19mobSacB. This work was supported by the U.S. National Institutes of Health (GM077596 to W.A.V.D.D. and W.W.M.).

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