Identification and Characterization of the PyridomycinBiosynthetic Gene Cluster of Streptomyces pyridomyceticusNRRL B-2517*□S

Received for publication, August 29, 2010, and in revised form, March 20, 2011 Published, JBC Papers in Press, March 22, 2011, DOI 10.1074/jbc.M110.180000

Tingting Huang ( ), Yemin Wang ( ), Jun Yin ( ), Yanhua Du ( ), Meifeng Tao ( ),Jing Xu ( ), Wenqing Chen ( ), Shuangjun Lin ( )1, and Zixin Deng ( )From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao TongUniversity, 1954 Huashan Road, Shanghai 200030, China

Pyridomycin is a structurally unique antimycobacterialcyclodepsipeptide containing rare 3-(3-pyridyl)-L-alanine and2-hydroxy-3-methylpent-2-enoic acid moieties. The biosyn-thetic gene cluster for pyridomycin has been cloned andidentified from Streptomyces pyridomyceticus NRRL B-2517.Sequence analysis of a 42.5-kb DNA region revealed 26 putativeopen reading frames, including two nonribosomal peptide syn-thetase (NRPS) genes and a polyketide synthase gene. A specialfeature is the presence of a polyketide synthase-type ketoreduc-tase domain embedded in an NRPS. Furthermore, we showedthat PyrA functioned as an NRPS adenylation domain that acti-vates 3-hydroxypicolinic acid and transfers it to a discrete pep-tidyl carrier protein, PyrU, which functions as a loadingmodulethat initiates pyridomycin biosynthesis in vivo and in vitro. PyrAcould also activate other aromatic acids, generating three pyr-idomycin analogues in vivo.

Formore than half of the twentieth century, natural productsand derivatives thereof have been essential products of thepharmaceutical industry because of the diversity of their struc-tures and biological activities (1). Many of these natural prod-ucts, such as cyclosporins, vancomycin, erythromycin, andFK506, are synthesized by multifunctional megasynthasemolecular “assembly lines” (2–5). There are two types ofmegasynthases: nonribosomal peptide synthetases (NRPSs)2and polyketide synthases (PKSs). They are composed of multi-ple large peptides, each ofwhich contains several catalyticmod-ules. They assemble in a stepwise fashion two distinct classes ofsecondary metabolites. The precursors, amino acids and shortcarboxylic acids, are selected and attached as thioesters to the

long phosphopantetheinyl arms of carrier proteins. They arethen linked by the condensation (C) domains inNRPSs via pep-tide bonds or by ketosynthase (KS) domains of PKSs that formC–C bonds by Claisen-like condensation. The mature chain isusually released from the synthase by cyclization or hydrolysiscatalyzed by a thioesterase (TE) domain in the terminal NRPSor PKS modules of the assembly line.NRPSs and PKSs are remarkably similar in their architec-

tures, which made it possible for nature to evolve PKS/NRPShybrid systems that combine NRPSs and PKSs in the sameassembly line and are capable of incorporating both carboxylicacids and nonproteinogenic amino acids into the final prod-ucts. Thus, the NRPS/PKS hybrid biosynthetic paradigm gen-erates enormous structural diversity (6, 7).Pyridomycin is an antimycobacterial antibiotic produced by

Streptomyces pyridomyceticus NRRL B-2517 (Fig. 1) (8). Pyr-idomycin is an unusual 12-membered ring depsipeptide com-posed of four moieties in the following order: N-3-hydroxypi-colinyl-L-threonine, 3-(3-pyridyl)-L-alanine, propionic acid,and 2-hydroxy-3-methylpent-2-enoic acid, which is probablyepimerized from �-keto-�-methylvaleric acid (9, 10). To thebest of our knowledge, it is the only known depsipeptide thatcontains an enolic acid. Isotope labeling studies (11) indicatedthat the biosynthesis of pyridomycin might involve theassembly of the backbone by a hybrid NRPS/PKS systemusing 3-hydroxypicolinic acid (3-HPA) as the starter unit.Feeding experiments confirmed that L-Asp is a precursor ofthe 3-hydroxypicolinyl moiety, whereas lysine was not incor-porated into the compound, indicating that the formation ofthe 3-hydroxypicolinyl moiety follows a different pathwayfrom that involved in the biosynthesis of streptogramin Bantibiotics, such as pristinamycin I, virginiamycin S (Fig. 1),and etamycin, which also use 3-HPA as the starter unit (12–14). Chemical synthesis schemes have been devised for pyr-idomycin (15), but little was known about the pathway ofpyridomycin biosynthesis.Here we report the cloning and sequencing of the pyridomy-

cin biosynthetic gene cluster within a 42.5-kb DNA region con-taining 26 open reading frames (ORFs) (Fig. 2A). At the centerof this region is a hybridNRPS/PKS system (NRPS/PKS/NRPS).The last NRPS contains an essential KR domain in an unusualposition. Based on targeted mutagenesis, in vitro enzyme stud-ies, and sequence analysis, we propose a putative pathway forpyridomycin biosynthesis. In vivo and in vitro characterization

* This work was supported by the Ministry of Science and Technology ofChina 973 and 863 programs and the National Science Foundation ofChina.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Methods,” Tables S1–S3, and Figs. S1–S9.

The nucleotide sequence(s) reported in this paper has been submitted to theGenBankTM/EBI Data Bank with accession number(s) HM436809.

1 To whom correspondence should be addressed. Tel.: 86-21-62932943; Fax:86-21-62932418; E-mail: linsj@sjtu.edu.cn.

2 The abbreviations used are: NRPS, nonribosomal peptide synthetase;3-HPA, 3-hydroxypicolinic acid; 4A2HBA, 4-amino-2-hydroxybenzoic acid;2,3-DHBA, 2,3-dihydroxybenzoic acid; 2-FBA, 2-fluorobenzoic acid; QTOF,quadrupole-time of flight; A domain, adenylation domain; PCP, peptidylcarrier protein; C domain, condensation domain; TE, thioesterase; PKS,polyketide synthase; KS, ketosynthase; KR, ketoreductase; ArCP, aryl carrierprotein; aa, amino acid(s); contig, group of overlapping clones.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 23, pp. 20648 –20657, June 10, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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of PyrA and PyrU demonstrated that PyrA and PyrU consti-tuted the loadingmodule that initiated the assembly of the pyr-idomycin backbone. Together with the observed broad sub-strate specificity of PyrA, these findings provide opportunitiesto generate pyridomycin derivatives with novel or enhancedbioactivities by rational engineering of the biosynthetic path-way or combinatorial biosynthesis. Given its special moleculararchitecture (9, 16), pyridomycin also offers an opportunity todiscover new chemistry for natural product biosynthesis.

EXPERIMENTAL PROCEDURES

Strains, Plasmids, and Culture Conditions—Strains and plas-mids used in the study are summarized in supplemental TablesS1 and S2. S. pyridomyceticus NRRL B-2517 and its derivativeswere cultivated at 30 °C in YEME liquid medium for growth ofmycelia, on COM medium (1% corn starch, 1% oat flour, 0.1%malt extract, 0.1% yeast extract, 0.1% tryptone, 1.2% agar, pH7.2) for sporulation, and on 2CM medium (17) (1% solublestarch, 0.2% tryptone, 0.1% NaCl, 0.2% (NH4)2SO4, 0.1%K2HPO4, 0.1% MgSO4, 0.2% CaCO3, 1.2% agar with 1 ml ofinorganic salt solution/liter and adjusted to pH 7.2) for conju-gation. Luria-Bertani (LB) broth and agar were used for cultur-ingEscherichia coli strains. All plasmid subcloning experimentswere performed inE. coliDH10Busing standard protocols (18).The final antibiotic concentrations used for selection of E. coliand Streptomyces were as follows: 5 �g/ml thiostrepton and 15�g/ml apramycin for S. pyridomyceticus; 30 �g/ml apramycin,50 �g/ml kanamycin, 100 �g/ml ampicillin, and 12.5 �g/mlchloramphenicol for E. coli.DNA Isolation and General Manipulations—DNA isolation

and manipulation in E. coli and Streptomyces were carried outfollowing the standard procedures (18, 19). PCR amplificationswere performedon aVeriti thermal cycler (AppliedBiosystems,Carlsbad, CA) using TaqDNA polymerase or KOD-plus highfidelity PCR polymerase. Synthetic PCR primers were obtainedfrom Invitrogen, and DNA sequencing was accomplished atShanghai Majorbio Biotech Co. Ltd. (Shanghai, China).Genomic Library Construction and Screening—A pOJ446-

derived genomic cosmid library of S. pyridomyceticus NRRLB-2517 was constructed according to a standard protocol (18,19). Recombinant clones were selected on LB agar plates sup-plemented with apramycin and screened by PCR using degen-erate primers for NRPS andKS that were designed according tothe CODENHOP algorithm (20).

DNA Sequence Analysis—ORFs were analyzed and identifiedusing the Frame Plot 3.0 beta online program (21), and thededuced proteins were compared with other known proteins inthe databases using the NCBI BLAST server (22). Multiplenucleotide sequence alignments and analysis were performedusing the BioEdit Sequence Alignment Editor (available on theWorld Wide Web) or Vector NTI Advance 11.0 (Invitrogen).The NRPS-PKS architecture was predicted by NRPS-PKS(available on the World Wide Web) (23).Inactivating PyrG-KR in S. pyridomyceticus NRRL B-2517 by

Point Mutation—The mutated sequence on pJTU4676PSY(ThioR; supplemental Fig. S1) was introduced into S. pyridomy-ceticus by interspecific conjugation. Thiostrepton-sensitive(ThioS) clones were selected and confirmed to contain thedesired chromosomal point mutation (HTT19PSY) using PCRand sequencing.Genetic Manipulation of pyrA and pyrU in S. pyri-

domyceticus—See the supplemental material.Pyridomycin Fermentation, Isolation, Feeding Experiments,

and HPLC-MS Analysis—See the supplemental material.Cloning of pyrU and pyrA—pyrU and pyrA were amplified

using KOD-plus high fidelity PCR polymerase (Toyobo) andcosmid 9A3 as template (primers listed in supplemental TableS3). The purified PCR products pyrU (255-bp NdeI/BamI frag-ment) and pyrA (1632-bp NdeI/EcoRI fragment) were ligatedinto pET28a digested with the same restriction enzymes togenerate plasmids pJTU4655 and pJTU4637. The correctsequences of pyrU in pJTU4655 and pyrA in pJTU4637 wereconfirmed by DNA sequencing.Construction of pJTU4655(S47A) and pJTU4652(S47A)—To

achieve themutation of serine to alanine at position 47 in PyrU,pJTU4655(S47A) and pJTU4652(S47A) were constructed bysite-directed mutagenesis using the primers PyrUS47AF andPyrUS47AR (supplemental Table S3). pJTU4655 andpJTU4652 served as templates. The mutations were verified byDNA sequencing. pJTU4655(S47A) was used for PyrU(S47A)overproduction, whereas pJTU4652(S47A) was used to com-plement the �pyrUmutant.Overproduction and Purification of Recombinant Proteins—

See the supplemental material.Phosphopantetheinylation of PyrU and PyrU(S47A)—The

in vitro assays for phosphopantetheinylation of PyrU orPyrU(S47A)were carried out in a 100-�l reaction containing 30�Mapo-PyrU, 0.5mMCoA, 3�MSfp, 10mMMgCl2, 2mMDTT,and 20mMTris�HCl, pH8.0, at 37 °C for 45min. Reactionswerestarted by adding Sfp (24) and quenched by flash freezing at�80 °C. The in vivo assays were performed by coexpression ofPyrU and PyrU(S47A) with Sfp in E. coli BL21 (DE3), followedby purification of the recombinant proteins.The assays were analyzed by HPLC on an Agilent HPLC

series 1100 with a ZORBAX 300SB-C18 column (4.6 � 250mm, 5 �m, 300 Å, Agilent), using a 10–90% (v/v) gradient ofacetonitrile/water containing 0.1% (v/v) trifluoroacetic acid(TFA) for 30 min at a flow rate of 0.5 ml/min and UV detectionat 280 nm.The LC-MS analyses were performed on a 6530 Accurate-

Mass QTOF spectrometer coupled to an Agilent HPLC 1200series (Agilent Technologies) that was developed for 30 min

FIGURE 1. Structures of natural products containing 3-hydroxypicolinicacid (boldface type). A, pyridomycin; B, pristinamycins IA (where R representsN(CH3)2) and IB (where R represents NHCH3) and virginiamycin S1 (where Rrepresents H).

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using a 5–95% linear gradient of acetonitrile/water containing0.1%TFA at a flow rate of 0.2ml/min.Datawere obtained in thepositive mode, and the mass scan range was set between 600and 2500 m/z. The resulting spectra were analyzed using thesoftware Mass Hunter, which calculated the masses of theintact proteins.Determination of Substrate Specificity of PyrA—PyrA activity

was measured by monitoring PPi release at 360 nm continu-ously for 30 min, using the EnzChek pyrophosphate assay kitaccording to themanufacturer’s instructions (MicroProbes) ona Synergy 2 multimode microplate reader (BioTek). PyrA (1.9�g and 0.3 �M) was used to determine the initial velocity with3-HPA or its analogs (4 mM) as the substrates. The standardcurve was created using the pyrophosphate standard from thekit. Reactions were carried out in triplicate with boiled PyrA ascontrol. Kinetic analysis of PyrA for 3-HPA, 2,3-dihydroxyben-zoic acid (2,3-DHBA), and 4-amino-2-hydroxybenzoic acid(4A2HBA) were performed by varying each substrate concen-tration (0.01–0.32 mM 3-HPA, 0.4–4.8 mM 2,3-DHBA, and0.4–4.0 mM 4A2HBA) in the presence of 2 mM ATP. The reac-tions were initiated, adding 1.2, 0.3, and 0.6 �M PyrA, respec-tively. The velocity was calculated based on the increase inabsorbance at 360 nm. The Michaelis-Menten equation wasfitted to plots of velocity of PPi release versus substrate concen-tration to extract values for Km and kcat using the programGraphPad Prism 5.Acylation of Holo-PyrU—Loading of 3-HPA and its analogs

onto holo-PyrU catalyzed by PyrA was performed in standardreactions containing 50 mM Tris�HCl (pH 8.0), 10 mMMgCl2, 2mM DTT, 5 mM ATP, 2 mM substrate, 5 �M PyrA, and 30 �M

holo-PyrU. Reactions were initiated by the addition of PyrA,incubated at 37 °C for 30min, and quenched by flash freezing at�80 °C. After centrifugation, the clarified supernatant was sep-arated by HPLC, and the new peak for candidate enzymaticproducts was collected and concentrated using a SavantSPD111V SpeedVac concentrator (Thermo Scientific). Theidentities of products were determined by HPLC and QTOFMS analysis as for the analysis of PyrU.

RESULTS

Cloning, Sequencing, and Analysis of the pyr Gene Cluster—To identify the pyridomycin biosynthetic gene cluster, agenomic library of S. pyridomyceticus NRRL B-2517 was con-structed in pOJ446 (about 2,000 cosmid clones). DegeneratePCR primers (supplemental Table S3) designed according tothe conserved core regions A3 and A7 of NRPS adenylation (A)domains (23, 25) were used for the initial screening of the cos-mid library, andmore than 100Adomain positive cosmidswereisolated. These were then tested using degenerate primersdesigned according to the conserved regions that are unique toKS domains of hybrid NRPS/PKS (7). More than 20 cosmidshybridizing to both the A domain and the KS domain probeswere isolated. Restrictionmapping produced two separate con-tigs. In order to identify the contig containing the pyridomycinbiosynthesis gene cluster, additional degenerate primers spe-cific for conserved motifs in 3-HPA:AMP ligases (responsiblefor activation of 3-HPA in the biosynthesis of pristinamycin,virginiamycin, and etamycin) (13, 14, 26) were used. The

deduced protein sequence of the amplified fragment from con-tig 2 resembled the 3-HPA:AMP ligase from Streptomyces pris-tinaespiralis (supplemental Fig. S2). From this, we concludedthat contig 2 may be involved in pyridomycin biosynthesis. Toconfirm this hypothesis, a 20-kb sequence in cosmid 9A3 ofcontig 2 was replaced by a kan cassette. Thismutation was thenintroduced into the S. pyridomyceticus chromosome. Theresulting strain, HTT7, no longer produced pyridomycin (sup-plemental Fig. S3). Thus, cosmid 9A3 was sequenced to yield acontinuous 42.5-kb DNA region, the GC content of which is73.56%, typical for Streptomyces (19), and 26 ORFs were pre-dicted (Fig. 2 and Table 1).Assembly of the Pyridomycin Core—Among the 26ORFs, two

typical NRPS genes, pyrE and pyrG, and a PKS gene, pyrF, wereidentified, and their functional domains matched the chemicalstructure of the pyridomycin core (Fig. 2A).PyrE consists of two minimal NRPS modules and is most

similar (45% identity) to DhbF, involved in the biosynthesis ofthe catecholic siderophore bacillibactin from Bacillus subtilis(27). The two A domains of PyrE are very similar to known Adomains, and they feature all 10 highly conservedmotifs. Mod-ule 1 of PyrE is similar to the pristinamycin I synthetase 2,which forms a 3-hydroxypicolinic acid-threoninemoiety aswaspredicted for pyridomycin biosynthesis. Also, the 10 residues inthe aa binding pocket predict incorporation of threonine bymodule 1 (28), consistent with the chemical structure and thefeeding experiments (11). PyrEmodule 2 probably incorporates3-(3-pyridyl)-L-alanine, which is similar to theweakly predictedphenylalanine.PyrF is a minimal PKS module. The KS domain features the

highly conserved catalytic Cys-His-His triad (29). The KSdomain is most similar to typical NRPS/PKS hybrid KSs fromblmIII (36% identity) or epoD (34% identity) (30, 31). Theacyltransferase domain of PyrF contains the highly con-served active site GHSXG and is similar to methylmalonyl-CoA acyltransferases.Special features of the PyrG (2451 aa) architecture are the

location of a PKSKRdomain and twoAdomains. PyrG is there-fore anNRPS/PKS hybrid protein. TheC domain in PyrG prob-ably forms a C–O (ester) link as has been found in fumonisin(32) and antibiotic C-1027 (33).The KR domain in PyrG was predicted to be functional

because it contains a Rossmann fold for NAD(P)H binding andconserved Lys, Ser, and Tyr residues (34) (supplemental Fig.S4). Mutation of conserved active sites of the KR domainS163A/Y176F resulted in complete loss of pyridomycin pro-duction, and no intermediate product was detected in thesupernatant or in the mycelium of three independent mutantclones. Analysis of the aa binding pockets of the two NRPS Adomains gave no firm prediction for the type of extender unit.The PyrG-A1 domain lost the conserved A3 motif, which iscritical for adenylate formation, whereas the A6 and A8 motifsin PyrG-A2 are not conserved (35) (supplemental Fig. S5). ATEdomain at the C-terminal end of PyrG is probably responsiblefor lactone formation, as shown in Fig. 2A.Biosynthesis of the Pyridyl Moieties—PyrB is an L-lysine

2-aminotransferase similar to VisA (61% identity and 70% sim-ilarity) involved in 3-HPA formation for virginiamycin biosyn-

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thesis in Streptomyces virginiae (14, 36) and to NikC (56% iden-tity and 69% similarity) that catalyzes the initial reaction forconverting lysine to the pyridyl residue in nikkomycin D inStreptomyces tendae (37). However, L-aspartic acid instead ofL-lysine (along with glycerol or pyruvic acid) was incorporatedinto the two pyridyl residues in pyridomycin. This suggestedthat pyrB should not be involved in the pyridomycin biosynthe-sis and that the formation of the pyridyl residues may followeither theNADbiosynthetic pathway (path a in Fig. 2B) (38, 39)or the aspartate family of amino acids biosynthetic pathway(path b in Fig. 2B) (40).

Five genes (pyrP to -T) are transcribed in the same orienta-tion and may constitute an operon (Table 1 and Fig. 2A). Theyinitially seemed to be involved in the biosynthesis of pyridylmoieties. PyrQ is a putative aspartate kinase (40). PyrP resem-bles 3-dehydroquinate synthase (41). The other genes encodeoxidoreductases. However, the inactivation of all of these genesdid not affect pyridomycin production (supplemental Tables S2and S3). These findings indicated that the biosyntheses of thepyridyl moieties would follow path a in Fig. 2B, using somegenes from the primary metabolism.

Unknown or Tentative Role in Pyridomycin Biosynthesis—Immediately downstream of the NRPS/PKS genes, pryH to -Nmay form an operon (Fig. 2A and Table 1). PyrH (71 aa) is 74%identical to the MbtH-like protein from Streptomyces fungici-dicus, and it contains the three conservedTrp residues thatmaybe important for moderating protein-protein interactions (42,43). Similar proteins are integral parts of several NRPSs thatstimulate specific A domains (44). PyrJ is probably a histidinol-phosphate aminotransferase catalyzing the formation of theenol carboxylic acid moiety �-keto-�-methylvaleric acid (mod-ule 4, Fig. 2). PyrI, -K, -L, and -M are putative oxidoreductases(cytochrome P450 or dehydrogenases), and PyrN is similar toan esterase. The roles of these enzymes in pyridomycin biosyn-thesis remain unclear.pyrC and pyrD, located upstream of NRPS/PKS genes,

encode a flavin-dependent oxidoreductase and a sarcosine oxi-dase, respectively. Similar gene pairs of unknown functions(snaO (67% aa identity) and snaN (58% aa identity), and virN(65% aa identity) and virM (58% aa identity)) occur in the pris-tinamycin II (45) and in the virginamycin M biosynthesis geneclusters (14).

FIGURE 2. Organization of the pyridomycin biosynthetic gene cluster and model for the biosynthesis of pyridomycin. A, at the top are ORFs 1–5 and PyrAto -U predicted from the 42.5-kb DNA sequence. Horizontal lines below the ORFs indicate the multifunctional NRPS and PKS modules. Below the moduledesignations are the functional domains: NRPS adenylation domains (A, A1, and A2), peptidyl carrier protein (PCP), condensation domain (C), thioesterase (TE),PKS ketosynthase (KS), acyltransferase (AT), acyl carrier protein (ACP), and ketoreductase (KR). The broken arrow to pyridomycin indicates that two additionalenzymatic steps are required to form the hydroxyl at the position indicated by an asterisk and the double bond at the position indicated by the number sign.B, alternative putative biosynthetic pathways for pyridyl moieties. Solid arrows, confirmed reactions (39, 58); dashed arrows, predicted reactions.

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Genes Involved in Regulation and Self-resistance—Pyr3, aputative membrane protein, could be involved in a transportsystem for pyridomycin export and self-resistance. PyrOresembles TetR regulators containing a conserved helix-turn-helix DNA-binding domain (Pfam00440) (46). Theinactivation of pyr2 (HTT12; supplemental Table S1) had noeffect on pyridomycin production (confirmed by LC-MSanalysis; data not shown), and it may be outside the biosyn-thetic gene cluster.3-HPA Activation by PyrA and PyrU—The above analysis of

PyrE to -G foundno loadingmodule. PyrA (543 aa), upstreamofthe NRPS/PKS/NRPS locus, is a probable 3-HPA:AMP ligasebecause it is similar to SnbA involved in pristinamycin I biosyn-thesis by S. pristinaespiralis (68% identity) andVisB involved invirginamycin S biosynthesis by S. virginiae (65% identity) (14,26). Presumably, 3-HPA is activated by PyrA and incorporatedinto the pyridomycin assembly line (Fig. 2A). To confirm therole of pyrA in pyridomycin biosynthesis, an internal 1101-bpDNA fragment (encoding the entire catalytic triad) wasreplaced in the S. pyridomyceticus genome using the PCR tar-geting method (Fig. 3, A and B). HPLC and bioassay showedthat pyridomycin production was completely abolished in thepyrA deletion mutant HTT6, but it was restored to near wild-type level by in trans complementation by a full-length pyrAgene expressed from PermE* (promoter of the erythromycinresistance gene) in the integrating plasmid pJTU4637b (Fig. 3,Cand D).Next to pyrA but reading in the opposite direction is pyrU,

which encodes a putative carrier protein. PSI-BLAST of itsdeduced amino acid sequence showed 42% identity to isocho-rismatase from Vibrio harveyi HY01, 37% to enterobactin syn-thetase component B inAzotobacter vinelandiiDJ, and 36% to a

conserved hypothetical protein from S. pristinaespiralisATCC25486. Sequence alignmentwith homologous proteins revealedan LGXXS motif in PyrU, which is a conserved motif in carrierproteins (47) (supplemental Fig. S6). Thus, PyrU is proposed tofunction as a PCP for tethering 3-HPA. No similar protein hasbeen found among the pristinamycin I and virginamycin S bio-synthetic genes.To confirm that pyrU is critical to the biosynthesis of pyrido-

mycin, the �pyrUmutant HTT5 was constructed (Fig. 4,A andB) and lost the production of pyridomycin, as analyzed byHPLC.Complementation by introducing pJTU4655 containingpyrU constitutively expressed from the ermE* promoter par-tially restored pyridomycin production of HTT5 (Fig. 4C). Asexpected, the mutant gene pyrUS47A failed to restore normalpyridomycin production. The bioassay using Mycobacteriumsmegmatismc2155 indicated that a trace amount of pyridomy-cin was produced by HTT5, and this was confirmed by LC-MSanalysis ([M � H]�541.3 m/z, identical to the pyridomycinstandard; Fig. 4, C and D). All of these findings clearly demon-strated that the putative PCP PyrU was essential for pyridomy-cin biosynthesis and that the serine 47 residue is the active sitefor the proposed phosphopantetheinylation.PyrU Is a Peptidyl Carrier Protein for Loading 3-HPA in Vivo

and in Vitro—To test whether PyrU functions as a PCP forloading 3-HPA, His6-apo-PyrU, His6-holo-PyrU, and, as a con-trol, the presumably inactive mutant His6-PyrUS47A wereoverproduced separately in E. coli (supplemental Fig. S7).

Phosphopantetheinylation in vivo was tested by coexpres-sion of PyrU and Sfp (B. subtilis phosphopantetheinyl transfer-ase expressed from pSV20) (48). Expression of PyrU withoutSfp was used as a control. Protein purification and HPLC anal-ysis showed that coexpression produced a new peak at 27.8min

TABLE 1ORFs of the pyr gene cluster, closest homologues, and proposed functions

ORF Sizea Homologous protein, species Identity/Similarity Accession number Proposed functionb

aa %Pyr1 49 Ccel_0989, Clostridium cellulolyticm H10 44/68 YP_002505331 FkbH-like proteinPyr2 246 SCO7266, Streptomyces coelicolor A3(2) 55/68 NP_631322 KetoreductasePyr3 334 Bthur0003_63860, Bacillus thuringiensis serovar thuringiensis

str. T0100129/53 ZP_04137149 Transport/self-resistance

Pyr4 210 Shewmr7_2680, Shewanella sp. MR-7 33/47 YP_738722 UnknownPyr5 72 SSDG_07478, S. pristinaespiralis ATCC 25486 71/87 EFH32214 UnknownPyrB 418 SSDG_07479, S. pristinaespiralis ATCC 25486 66/75 EFH32215 AminotransferasePyrA 543 SSDG_07480, S. pristinaespiralis ATCC 25486 68/76 EFH32216 3-Hydroxypicolinic acid:AMP ligasePyrU 84 SSDG_05103, S. pristinaespiralis ATCC 25486 36/59 ZP_05014213 Phosphopantetheine bindingPyrC 384 SSDG_05104, S. pristinaespiralis ATCC 25486 67/76 ZP_05014214 OxidoreductasePyrD 381 SSDG_05106, S. pristinaespiralis ATCC 25486 60/71 ZP_05014216 OxidasePyrE 2147 SgriT_010100025868, Streptomyces griseoflavus Tu4000 49/61 ZP_05541578 Dimodular NRPS (C-A-T-C-A-T)PyrF 1411 Ava_1612, Anabaena variabilis ATCC 29413 38/55 YP_322130 PKS (KS-AT-ACP)PyrG 2451 SAML0376, S. ambofaciens ATCC 23877 45/55 CAJ89363 NRPS (C-A1-A2-KR-T-TE)PyrH 71 Sros_3469, S. fungicidicus 63/74 YP_003339148 MbtH-like proteinPyrI 402 SBI_09258, Streptomyces bingchenggensis BCW-1 52/66 ADI12376 Cytochrome P450PyrJ 372 SBI_05317, S. bingchenggensis BCW-1 65/76 ADI08437 AminotransferasePyrK 173 ROP_53460, Rhodococcus opacus B4 53/65 YP_002782538 OxidoreductasePyrL 402 SSAG_05882, Streptomyces sp. Mg1 74/83 ZP_05001560 DehydrogenasePyrM 488 nfa46290, Nocardia farcinica IFM 10152 53/64 YP_120844 DehydrogenasePyrN 239 SBI_07222, S. bingchenggensis BCW-1 49/60 ADI10342 EsterasePyrO 212 SAV_2270, Streptomyces avermitilisMA-4680 45/64 NP_823446 TetR family regulatorPyrP 370 Francci3_4206, Frankia sp. CcI3 47/66 YP_483283 3-Dehydroquinate synthasePyrQ 454 SCAB_41651, Streptomyces scabiei 87.22 39/56 YP_003489785 Aspartate kinasePyrR 408 MXAN_4919,Myxococcus xanthus DK 1622 37/53 YP_633075 Cytochrome P450PyrS 513 BTH_II2161, Burkholderia thailandensis TXDOH 43/59 YP_440349 DehydrogenasePyrT 497 nfa32650, Nocardia farcinica IFM 10152 68/82 YP_119476 Aldehyde dehydrogenase

a Number of amino acids in the ORF predicted by Frame Plot 3.0.b Functions of the most similar proteins from the NCBI database and from the predicted conserved motifs. NRPS and PKS domains are abbreviated as follows: A, adenyla-tion; C, condensation; T, thiolation; KS, ketoacyl synthase; AT, acyltransferase; ACP, acyl carrier protein; KR, ketoreductase; TE, thioesterase.

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compared with the peak eluting at 28.3 min from PyrU alone(Fig. 5A, a and b). The identity of the two peaks was confirmedby ESI-QTOF-MS analysis, giving 11,294.90 Da (calcd:11,294.77 Da for apo-PyrU) and 11,635.21 Da (calcd: 11,635.86Da for holo-PyrU), which is a �340 mass shift, consistent witha 4�-phosphopantetheine cofactor covalently attached to theSer residue of apo-PyrU (Fig. 5B) (49). However, PyrU with theS47A mutation coexpressed with pSV20 only gave one peak at

28.8 min on HPLC analysis, showing that it was inactive (Fig.5A, d and e).Subsequently, we monitored the incorporation of the

4�-phosphopantetheine cofactor in vitro.When incubated withCoA and Sfp, apo-PyrU was quantitatively converted to holo-PyrU (Fig. 5A, c), as demonstrated by HPLC analysis, whereasno change was observed for PyrUS47A. Together, the data pro-vide direct evidence that PyrU is a PCPwith an active site Ser47.

FIGURE 3. Construction and fermentation of the �pyrA mutant HTT6. A, schematic representation of the pyrA gene replacement in HTT6. B, confirmation ofthe inactivation of pyrA by insertion of an apramycin resistance-oriT cassette (aac(3)IV � oriT) by PCR using the primers pyrAidx1-f and pyrAidx1-r; the wild typegave a 2710 bp band, whereas all mutants yielded the expected 2994 bp band. M, 1 Kb plus DNA ladder (Invitrogen). C, HPLC profiles. a, pyridomycin standard;b– d, extracts from the wild-type strain (WT), non-producing mutant HTT6, and HTT6 complemented in trans by a functional pyrA gene. The slanted arrows pointto the pyridomycin peak. D, bioassay with M. smegmatis. a– d, equal amounts of the samples labeled as for the HPLC profiles.

FIGURE 4. Construction and fermentation of �pyrU mutant HTT5. A, schematic representation of the pyrU mutant HTT5 by double crossover gene replace-ment. B, conformation of the replacement of pyrU by PCR using the primers pyrU2NF and pyrU2BR; the wild-type gave a 255 bp band, whereas all mutants gavethe expected 1426 bp band. M, 1 Kb plus DNA ladder (Invitrogen). C, HPLC profiles of fermentation. a, pyridomycin standard; b– e, extracts from the WT strain,mutant HTT5, and HTT5 complemented by pyrU or pyrUS47A. The slanted arrows point to the pyridomycin peak. D, bioassay with M. smegmatis. a– d, equalamounts of the samples labeled as for the HPLC profiles. AU, absorbance units.

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Substrate Specificity of PyrA—To determine the substratespecificity in vitro, the 58.9-kDaN-terminallyHis6-taggedPyrAwas produced in E. coli (supplemental Fig. S8). The reactionvelocity of PPi release fromATP, catalyzed by PyrA with differ-ent aromatic acids and amino acids at 4mMconcentrations, wasdetermined using a continuous spectrophotometric assay (see“Experimental Procedures”). The results depicted in Fig. 6Ashowed that 2,3-DHBA and 4A2HBA were activated with evenhigher reaction velocity than 3-HPA. TheKm and kcat values forthese three substrates were determined and are shown in Fig.6C. Although higher turnover numbers (kcat) were indeedfound for 2,3-DHBAand 4A2HBA, the catalytic efficiency (kcat/Km) of PyrA for 3-HPAwas 22- and 40-fold higher than that for2,3-DHBA and 4A2HBA, respectively. Taken together, theresults are consistent with the assignment of 3-HPA as thenative substrate of PyrA. However, the capability of PyrA toaccept a range of different substrates if supplied at sufficientconcentration offers the prospect to generate pyridomycinderivatives by feeding alternative starter units to a strain thathas lost by mutation the ability to synthesize 3-HPA.In Vitro Reconstitution of the Loading Module for the Pyrido-

mycin Biosynthesis—The functions and the location of PyrAand PyrU led to the expectation that PyrA may activate 3-HPAand tether it to PyrU, followed by transfer to the NRPS/PKSassembly line. To confirm this hypothesis, His6-holo-PyrU,His6-PyrA, and the substrate 3-HPAwere incubated in a buffercontainingMg2�, DTT, and ATP. HPLC analysis and high res-olution ESI-QTOF-MS showed a new peak at m/z 11,756.32Da, consistent with the expected 3-HPA-holo-PyrU (calcd:11,756.87 Da) (Fig. 7, a and b).The 3-HPA analogs 4A2HBA and 2-fluorobenzoic acid

(2-FBA) also yielded the expected products at m/z 11,770.37and 11,757.40 Da (calcd: 11770.89 and 11,757.87 Da, respec-tively) (Fig. 7, c and d). Therefore, PyrA tethers 3-HPA and

alternative aromatic molecules to PyrU, which acts as the load-ing module for pyridomycin assembly.Production of New Pyridomycin Analogues—Inspired by the

broad substrate specificity of PyrA and PyrU, we created newpyridomycin analogs by feeding picolinic acid, 2,3-DHBA,4A2HBA, 2-FBA, and 2-chlorobenzoic acid into the wild-typestrain. LC-QTOF-MS revealed three analogs derived from pic-olinic acid, 2,3-DHBA, and 4A2HBA (Table 2 and supplemen-tal Fig. S9). No new product was generated from 2-FBA or2-chlorobenzoic acid.

DISCUSSION

In this work, we report the cloning and characterization of a42.5-kb DNA fragment of S. pyridomyceticus NRRL B-2517,which contains a gene cluster that encodes the enzymes for theassembly of the core structure of the antimycobacterial antibi-otic pyridomycin.The gene cluster represents an unusual NRPS/PKS hybrid

system. PyrE, anNRPS elongationmodule containing twomin-imal modules (C-A-PCP), activates and tethers threonine and3-(3-pyridyl)-L-alanine to the PCPs of NRPS1 and NRPS2,respectively, and forms an amide bond. Next, PyrF, a typicalPKS elongation module, probably activates and transfers amethylmalonyl CoA to the PKS3 acyl carrier protein and elon-gates the chain.Surprisingly, the PKS3, PyrF, lacks a KR domain that was

thought to be responsible for the reduction of the �-keto group(*) to the hydroxyl group (*) of pyridomycin (Fig. 2A).A probably functional KR domain is embedded in the subse-

quent NRPS4 (PyrG). Based on the structural analysis of pyr-idomycin, the KR in PyrG was initially predicted to catalyze thereduction of the �-keto group (*) of the biosynthetic interme-diate transferred from PKS3. Disruption by nonpolar pointmutations should thus have produced a keto group at the posi-

FIGURE 5. Phosphopantetheinylation of PyrU. A, HPLC profiles for phosphopantetheinylation. a, apo-PyrU (�) from E. coli BL21(DE3)/pJTU4655; b, holo-PyrU(�) biosynthesized by coexpression of PyrU and Sfp; c, in vitro conversion of apo-PyrU to holo-PyrU catalyzed by Sfp (F); d, apo-PyrUS47A (�) from E. coliBL21(DE3)/pJTU4655(S47A); e, coexpression of PyrUS47A and Sfp. B, QTOF-MS analysis of apo-PyrU and holo-PyrU. The mass shift of 340 Da is consistent withthe phosphopantetheinyl moiety transferred from CoA to apo-PyrU. AU, absorbance units; amu, atomic mass units.

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tion in pyridomycin (indicated by asterisk in Fig. 2A). However,no product was detected, indicating that chain assembly wasprematurely aborted.Similar NRPS modules have been described for cereulide,

valinomycin, cryptophycin, and hectochlorin biosynthesis

(50–53). They all catalyze the reduction of the �-keto carboxylacid that is tethered directly to the PCP by a cognate A domain.As predicted from the in vitro biochemical investigation (54),the KR point mutant HTT19PSY (S163A/Y176F) did not pro-duce any pyridomycin intermediate. The conservative point

FIGURE 6. Substrate specificity of PyrA in vitro. A, activation of different acyl substrates (4 mM) by PyrA. PPi release was determined by a continuousspectrophotometric assay with 30-min reaction. Error bars, S.D. values from three independently performed experiments. B, structures of the tested substrates.C, determination of Km and kcat values for 3-HPA (left), 2,3-DHBA (middle), and 4A2HBA (right).

FIGURE 7. HPLC profiles for acyl-PyrU formation catalyzed by PyrA. a, control performed with PyrA (F) and holo-PyrU (�) without the acyl substrate; b,3-HPA-PyrU (�) formation with 3-HPA as a substrate; c, 4A2HBA-PyrU (ƒ) formation with 4A2HBA as a substrate; d, 2-FBA-PyrU (�) formation with 2-FBA as asubstrate. AU, absorbance units.

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mutations were unlikely to change the overall structure of theNRPS4. It therefore seemed likely that the KR domain needs tobe functional and is essential for pyridomycin production.Probably, �-keto-�-methylvaleric acid (derived from isoleu-

cine) is activated by the tandemA domains of NRPS4 and teth-ered to PCP. Then theKR domainmay reduce the�-keto groupof�-keto-�-methylvaleric acid to hydroxy, ready for ester bondformation catalyzed by the C domain (Fig. 2A). The TE domainthen detaches and circularizes the chain. This would generate ahypothetical precursor that needs to be reduced at the positionindicated by the asterisk in Fig. 2A and oxidized to form a dou-ble bond (#) in the structure of pyridomycin. The enzymes cat-alyzing these steps remain to be identified.The tandem A domains of PyrG may act together to activate

the substrate and tether it to PCP because PyrG-A1 lacks theconservedA3motif for adenylate formation, and theA6 andA8motifs in PyrG-A2 are not conserved. The TE domain at the Cterminus of PyrG was proposed to catalyze the cyclization ofthe mature pyridomycin linear chain to form the lactone.PyrE, PyrF, and PyrG constitute the hybrid NRPS/PKS that

synthesizes the pyridomycin ring structure. However, anenterobactin EntB-like loading module containing an ArL-ArCP didomain was still missing for the pyridomycin assemblyline (55).PyrA, a predicted 3-HPA:AMP ligase, was shown to linkATP

and 3-HPA, releasing PPi. 3-HPA:AMP ligases were proved tobe involved in the biosynthesis of streptogramin B antibiotics,such as pristinamycin I, etamycin, and virginiamycin S (12–14).Therefore, PyrA was envisioned to activate 3-HPA using ATP.Indeed, the in vivo and in vitro experiments proved that PyrAselected and activated 3-HPA in the presence of ATP, a func-tion that is normally performed by NRPS A domains.The search for a carrier protein identified PyrU, a small 84-aa

protein that has not been observed in the streptogramin B anti-biotics biosynthesis (14, 45). Although it shows low homologywith known PCPs or aryl carrier proteins (ArCPs), it featuresthe conserved LGXXS motif for phosphopantetheinylation(supplemental Fig. S6). Therefore, PyrU was proposed to func-tion as a PCP orArCP receiving the activated 3-HPA fromPyrA(55–57). To confirm the function of PyrU, the �pyrU mutantHTT5 was constructed. It almost completely lost pyridomycinproduction, which was restored by trans complementation,demonstrating the involvement of PyrU in pyridomycin bio-synthesis. To obtain clear evidence that PyrU functions as aPCP/ArCP for loading 3-HPA, PyrU and its mutant PyrUS47Awere overproduced and purified for biochemical characteriza-tion. Phosphopantetheinylation of PyrU but not of PyrUS47Awas observed in vivo and in vitro, clearly identifying PyrU as aPCP/ArCP.With the characterized PyrA and PyrU in hand, we

successfully reconstituted the loading module for pyridomycinassembly in vitro.

Pyridomycin and pristinamycin I use the same two initialbuilding blocks. A predicted PCP gene (pyrU orthologue;SSDG_07480; Table 1) was found in the S. pristinaespiralisATCC 25486 genome sequence, outside the pristinamycin bio-synthetic gene cluster (45). We thus predict that the separatePCP may participate in the biosynthesis of pristinamycin I andother streptogramin B compounds.Streptogramin B and pyridomycin contain 3-HPA starter

units. The streptogramin 3-HPA is derived from lysine (14, 36,37), but the labeling experiment indicated that both pyridylmoieties of pyridomycin originate from L-aspartic acid, glyc-erol, and/or pyruvate, but lysine is not incorporated (11). Pyri-dyl ring formation from aspartate is known from the primarymetabolismNAD biosynthetic pathway (39) (path a in Fig. 2B).The three initial steps of path b in Fig. 2B (aspartate kinase,aspartate semialdehyde dehydrogenase, and dihydropicolinatesynthase) are also used in the biosynthesis of lysine (40). Forpyridyl ring formation, dehydrogenation produces pyridine2,6-dicarboxylic acid (58), which can be envisioned to be con-verted to picolinic acid by decarboxylation. However, inactiva-tion of the predicted aspartate kinase pyrQ did not reduce pyr-idomycin production. We therefore propose that both pyridylmoieties are synthesized by the reactions shown in Fig. 2, patha, similar to NAD biosynthesis in the primary metabolism andthe shikimate pathway (38).PyrA activated a series of aromatic acids, including two aro-

matic amino acids, and transferred them to PyrU. Precursorfeeding of S. pyridomycetus yielded three pyridomycin analogs(Figs. 6 and 7 and supplemental Fig. S9), but no product wasgenerated from 2-chlorobenzoic acid or 2-fluorobenzoic acid.A combination of rational engineering of the biosynthetic path-way and precursor feeding will provide opportunities to pro-duce novel pyridomycin derivatives. Thiswork sets the stage forongoing in depth investigations of pyridomycin biosynthesis.

Acknowledgments—We are grateful to Dr. Tobias Kieser for criticalreading of the manuscript and valuable comments and Prof. LutzHeide for helpful discussions. We thank Dr. Wen Liu from the Shang-hai Institute of Organic Chemistry, Chinese Academy of Sciences, forkindly providing plasmid pSV20.

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Chen, Shuangjun Lin and Zixin DengTingting Huang, Yemin Wang, Jun Yin, Yanhua Du, Meifeng Tao, Jing Xu, Wenqing

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

doi: 10.1074/jbc.M110.180000 originally published online March 22, 20112011, 286:20648-20657.J. Biol. Chem. 

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Supplemental material:

  http://www.jbc.org/content/suppl/2011/03/22/M110.180000.DC1

  http://www.jbc.org/content/286/23/20648.full.html#ref-list-1

This article cites 55 references, 12 of which can be accessed free at

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