The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp [perhydrolase]and bpsD [nonribosomal peptide synthetase D] genes, which areinvolved in the synthesis of the balhimycin building block ß-hydroxytyrosine[ß-HT] . Reverse transcriptase experiments revealedthat bhp, bpsD, and oxyD form an operon . oxyD was inactivatedby an in-frame deletion, and the resulting mutant was unableto produce an active compound . Balhimycin production could berestored [i] by complementation with an oxyD gene, [ii] in cross-feedingstudies using A . balhimycina JR1 [a null mutant with a blockin the biosynthesis pathway of the building blocks hydroxy-and dihydroxyphenylglycine] as an excretor of the missing precursor,and [iii] by supplementation of ß-HT in the growthmedium . These data demonstrated an essential role of OxyD in the formation pathway of this amino acid . Liquid chromatography-electrosprayionization-mass spectrometry analysis indicated the biosynthesisof completely chlorinated balhimycin by the oxyD mutant whenculture filtrates were supplemented with nonchlorinated ß-HT.In contrast, supplementation with 3-chloro-ß-HT didnot restore balhimycin production . These results indicated that the chlorination time point was later than the stage of free ß-HT, most likely during heptapeptide synthesis.

 
In the past several decades, the glycopeptide antibiotic vancomycin became the antibiotic of last resort for the treatment of infections caused by multiresistant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus strains [37] . However, the occurrenceof vancomycin-resistant bacteria [3] and the expected increasein resistance may limit the medical use of even vancomycin inthe near future . The search for new glycopeptide antibioticsis essential to overcome this problem . One strategy for obtainingnew glycopeptides is to genetically manipulate the producer strains . This approach requires a molecular understanding of glycopeptide biosynthesis.

In order to study the biosynthesis of glycopeptide antibioticsand the functions of the relevant genes [6, 22, 31], we chosethe balhimycin producer strain Amycolatopsis balhimycina DSM5908[36] as a model system . A . balhimycina belongs to the orderof Actinomycetales and was formerly described as Amycolatopsis mediterranei [8, 18] . It was isolated from an Indian soil sampleoriginating from the Himalayas [18]. A . balhimycina shows thetypical growth characteristics of actinomycetes and forms anorange substrate mycelium but no spores when cultivated on solidmedia [20] . The in vitro and in vivo activities of balhimycinare comparable to those of vancomycin [34], but balhimycin showsa slight increase in antibiotic activity toward anaerobic bacteria[for example, clostridia] [8].

The chemical structure of vancomycin-type antibiotics [Fig. 1] such as balhimycin is based on a central heptapeptide core. This peptide core contains five aromatic amino acids . In the case of vancomycin and balhimycin, the nonproteinogenic aminoacids 4-hydroxyphenylglycine [HPG; positions four and five], 3,5-dihydroxyphenylglycine [DPG; position seven], and ß-hydroxytyrosine [ß-HT; positions two and six] are incorporated . Thesearomatic acid side chains are linked to each other to form twodiaryl ether rings and one biaryl ring, and the aglycone thusformed is modified by sugar substituents . The formation pathwaysof DPG and HPG have been studied in detail [11, 15, 23] . Furthermore,investigations of the synthesis of ß-HT revealed theparticipation of the perhydrolase Bhp [25] and the nonribosomalpeptide synthetase module BpsD [27] . As an additional modificationof the peptide core, both ß-HT residues of balhimycinare chlorinated [Fig . 1] . In general, chlorine atoms as wellas glycosyl groups have a strong influence on the antibioticactivities of glycopeptides [12, 13, 19], most likely by stabilizing the dimerization of these compounds [4, 12, 16].

 
Recently, the NADH/FAD-dependent halogenase BhaA was identifiedas essential for the chlorination reaction of both ß-HTresidues, at positions 2 and 6 of the glycopeptide aglycone[25] . However, the substrate of BhaA and therefore the chlorinationtime point during balhimycin biosynthesis remained unclear.

Here we report that the putative P450 monooxygenase OxyD, together with perhydrolase Bhp and the nonribosomal peptide synthetaseBpsD, is required for the formation of the nonproteinogenicamino acid ß-HT . In addition, we present evidencethat the chlorination of the ß-HT residues does nottake place either during precursor synthesis or at the aglycone.

 
Bacterial strains and plasmids. The strains and plasmids used for this study are listed in Table1.

 
Media and culture conditions. Escherichia coli strains were grown in Luria broth [28] supplementedwith 150 µg of ampicillin ml^–1 or 100 µg ofapramycin ml^–1 when necessary to maintain plasmids . A.balhimycina strains were grown in R5 medium [14] at 30°C.Liquid and solid media were supplemented with 50 µg oferythromycin ml^–1 or 50 µg of apramycin ml^–1 to select for strains carrying integrated antibiotic resistancegenes.

Cultivation of OP090 in the presence of ß-HT derivatives. OP090 [Table 1] was incubated under standard conditions in 20 ml of R5 medium [14] . After 24 h of growth, ß-HT orchloro-ß-HT [CHT] was added [1 mg/ml] . The supernatants were harvested at different time points, and 20 µl ofeach was used to determine the production of balhimycin in abioassay with Bacillus subtilis.

Preparation of A . balhimycina RNA. A . balhimycina was cultivated in 100 ml of R5 medium for 3 days.The cells were then harvested and shock frozen at –70°C.An aliquot was resuspended in 100 µl of P buffer [32]containing 10 mg of lysozyme and then incubated for 7 min at37°C . The RNA was extracted by use of an RNeasy mini kit[Qiagen, Hilden, Germany] according to the manufacturer's instructions.

RT-PCR analysis. RNA prepared from A . balhimycina was treated with 3 U of RNase-freeDNase I [Promega, Madison, Wis.] and precipitated accordingto standard protocols [28] . The RNA concentration was photometricallydetermined with a Genequant fixed-wavelength photometer [Pharmacia,Freiburg, Germany] . Reverse transcription [RT] reactions wereperformed by use of an Omniscript RT kit [Qiagen] accordingto the manufacturer's instructions . The primers used for RTof the bhp-bpsD and bpsD-oxyD overlapping mRNA sequences werecotraorfYTGA [5'-TCAGCGTGGTGGTCCCCATC-3'] and cotraoxyDTGA [5'-CCAGAAGCCGGAGGGGGAAC-3'], respectively . PCRs were carried out in a programmable thermal controller [MJ Research, Inc., La Jolla, Calif.] under the following conditions: initial denaturation [95°C for 2 min]; 25 cyclesof denaturation [95°C for 20 s], annealing [60°C for30 s], and polymerization [72°C for 40 s]; and finally,an additional polymerization step [72°C for 7 min] . EachPCR mixture [25 µl] contained a 1-µl aliquot ofRT reaction product, 100 pmol of each primer, deoxyribonucleoside5'-triphosphates at a final concentration [each] of 20 µM[DNA polymerization mix; Pharmacia], 10x reaction buffer [Qiagen],5x Q solution [Qiagen], and 3.5 U of Taq DNA polymerase [Qiagen]. The following oligonucleotide primer pairs were used: cotraorfYATG [5'-AGGAGCTGGCCGCCGTGATC-3'] and cotraorfYTGA [5'-TCAGCGTGGTGGTCCCCATC-3'], for amplification of the bhp-bpsD overlapping fragment, and cotraoxyDATG [5'-CGGAAGTGCTCGGTGTCAGC-3'] and cotraoxyDTGA [5'-CCAGAAGCCGGAGGGGGAAC-3'],for amplification of the bpsD-oxyD overlapping fragment . ThePCR products were analyzed by agarose gel electrophoresis [1.0%].

Preparation and manipulation of DNA. The methods used for the isolation and manipulation of DNA weredescribed by Sambrook et al . [28] and Hopwood et al . [14] . PCR fragments were isolated from agarose gels with a Qiaquick kit [Qiagen] . Restriction endonucleases were obtained from various suppliers and were used according to their specifications.

PCR protocols for amplification of fragments frOP3, frOP4, and the oxyD gene and for characterization of OP090k. PCRs were performed with a programmable thermal controller [MJResearch, Inc.] . Each PCR mixture [100 µl] contained 100pmol of each primer, 1.0 µg of template DNA [cosmid 16.1],deoxyribonucleoside 5'-triphosphates at a final concentration[each] of 20 µM [DNA polymerization mix; Pharmacia], 10xreaction buffer [Qiagen], 5x Q solution [Qiagen], and 3.5 U of Taq DNA polymerase . Dimethyl sulfoxide [Stratagene] was added to the reaction mixture at a final concentration of 3% to enhance the specificity of hybridization . For amplification of the fragments frOP3 and frOP4, which are part of the deletion plasmid pOP2 [see below], the following PCR conditions were used: initial denaturation [95°C for 2 min] before the addition of thepolymerase; 30 cycles of denaturation [95°C for 20 s], annealing[68°C for 30 s], and polymerization [72°C for 1 min45 s]; and an additional polymerization step [72°C for 7min] at the end . The primers used were as follows: for amplificationof the fragment frOP3, XI [5'-GGTCTGATCGCCCGCGGTTACCTGCACCGGCCG-3']and XII [5'-GGTCTAGAGATATCGGTGTGCGCCTGCCGCGGGGTCATCC-3']; andfor amplification of the fragment frOP4, XIII [5'-GGGATATCGACGACCCGGACACCTTCCTGCCCGG-3']and XVI [5'-GGGATATCGCACGTTCGTCGACCGCAGGTCGTCC-3'] . For amplificationof the oxyD gene, the annealing step was done at 60°C for30 s and the polymerization step was done at 72°C for 1min 30 s . The sequences of the primers were as follows: oxyDlow, 5'-GAGATCTTGGAGACCCTGATGCAGACG-3'; and oxyDup, 5'-GAGATCTGGTCAGCGCCCGGTGAACC-3'. For confirmation of the integration of pSET-oxyD [see below] into the genome of OP090k outside of the oxyD locus, an annealing temperature of 55°C [30 s] was used . The polymerizationstep was done at 72°C for 30 s . The sequences of the primers,which amplified a 1,078-bp fragment of the balhimycin gene cluster containing the entire oxyD gene construct, were as follows:oxyDex1, 5'-GAGGACAGCTTCTTCGAGGTCG-3'; and oxyDex2, 5'-CGCATCAACGGTGTCAGCTT-3'.

Construction of plasmids pOP2 and pSET-oxyD. Plasmids were constructed for an internal deletion of the P450monooxygenase gene oxyD [pOP2] as well as for complementationof the oxyD deletion mutant strain A . balhimycina OP090 [pSET-oxyD].

[i] pOP2. The 1,300-bp fragment frOP3, including a sequence encoding 188amino acids of the N terminus of the P450 monooxygenase geneoxyD [1,191 bp], and the 1,287-bp fragment frOP4, includinga sequence encoding 76 amino acids of the C terminus of oxyD,were ligated into the EcoRV site of the vector pJOE890, resultingin the plasmids pJOEOP3 and pJOEOP4, respectively . frOP3 wasthen ligated as an XbaI fragment into the single XbaI site of the vector pSP1, resulting in the plasmid pSPOPb . To obtainthe plasmid pOP2, containing a partly deleted oxyD gene, we ligated frOP4 as an EcoRV fragment into the EcoRV site of pSPOPb.

[ii] pSET-oxyD. A 1,217-bp fragment consisting of oxyD and its ribosomal bindingsite was ligated as a blunt-ended fragment into the vector pJOE890and then integrated into the BamHI cleavage site of the vectorpUC18ermEp1 as a BglII fragment, resulting in the plasmid pUC-oxyD.The ermE*p-oxyD expression construct was then ligated as anEcoRI-XbaI fragment into the single EcoRI-XbaI site of the vectorpSET152, resulting in the complementation plasmid pSET-oxyD.

Cross-feeding studies with different A . balhimycina strains. To investigate whether different null mutant strains were blockedin the same biosynthetic pathway, we performed cross-feeding experiments . For these studies, the strains of interest wereplated on an R5 agar plate, with a small cell-free region [approximately0.5 cm] left between them . After 5 days of incubation [30°C],an agar strip containing both mutants was cut out and analyzedin a bioassay with B . subtilis to determine whether the diffusionalexchange of accumulated intermediates restored the balhimycinproduction ability of the tested null mutants.

Determination of ß-HT and CHT uptake by A . balhimycina. To determine the uptake of ß-HT and CHT by A . balhimycina, we measured the concentrations of the amino acids in the medium at different time points by reversed-phase high-performanceliquid chromatography [HPLC] . The cell-free supernatant [sampleinjection volume, 20 µl] was separated at a flow rateof 2 ml/min on a Nucleosil C₁₈ column [12.5 cm by 0.4 cm by5 µm] via a gradient elution using the ThermoSeparationspectrum system [pump, model P200; automatic probe injector,model AS3000; UV detector, model UV3000HR; Thermo Request Systems,Egelsbach, Germany] . The following gradient was used: at t =0 min, 100% A; at t = 10 min, 80% A and 20% B; at t = 13 min,100% B; at t = 16 min, 100% A [solvent A, 0.1% phosphoric acid;solvent B, acetonitrile].

Determination of balhimycin biosynthesis by HPLC-ESI-MS. Balhimycin production was determined with bioassays using cell-free supernatants of Amycolatopsis strains grown on R5 medium, with B . subtilis ATCC 6633 as a test organism [14] . Investigationsof the balhimycin variants in culture broth were performed byHPLC-electrospray ionization-mass spectrometry [HPLC-ESI-MS].Culture broth was prepared by centrifugation and filtrationto obtain particle-free samples . LC-ESI-MS experiments wereperformed on a Bruker Esquire 3000+ instrument coupled to an Agilent 1100 HPLC system [Bruker-Franzen, Bremen, Germany]. Separations were performed on a Nucleosil C₁₈ column [2 mm by 100 mm by 5 µm] [Grom, Herrenberg, Germany] at a flowrate of 200 µl min^–1 . The following gradient wasused: at t = 0 min, 95% A and 5% B; at t = 1 min, 83% A and17% B; at t = 15 min, 80% A and 20% B; at t = 17 min, 100% B [solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic acid in acetonitrile].

Nucleotide sequence accession number. The nucleotide sequences of the balhimycin biosynthetic genesreported in this paper are available from the EMBL data libraryunder accession number Y16952.

 
The P450 monooxygenase gene oxyD is part of an operon including the genes bhp and bpsD. In the balhimycin biosynthetic gene cluster, altogether fourgenes [oxyA to -D] have been identified whose gene productsshow significant similarities to P450 monooxygenases . OxyA-,-B, and -C show higher sequence homologies to each other [41to 46% similarity; 55 to 64% identity] than to OxyD [26 to 31%similarity; 38 to 48% identity] . For the vancomycin producer,it has been shown that the homologous oxygenases are P450 monooxygenases[38] . OxyA, -B, and -C in A . balhimycina catalyze the cross-linkingsteps between the aromatic rings within the balhimycin peptidebackbone in a defined order [6] . In contrast, the function ofOxyD remained unclear . The genes oxyA to -C are clustered in a region approximately 12.7 kb upstream of oxyD, which lies directly downstream of the perhydrolase gene bhp and the gene bpsD, which codes for a nonribosomal peptide synthetase [NRPS]. Since Bhp and BpsD are involved in ß-HT formation[25, 27], it was assumed that OxyD also participates in this pathway . No termination signals were detectable in the intergenic DNA sequences of bhp, bpsD, and oxyD, indicating cotranscriptionof the three genes . In order to prove the operon structure ofthe bhp-bpsD-oxyD region, we performed an RT-PCR analysis . Usingprimer pairs corresponding to [i] the 3' region of bhp and the5' region of bpsD and [ii] the 3' region of bpsD and the 5'region of oxyD, we amplified [i] a bhp-bpsD overlapping fragment[346 bp; contains 155 bp of the bhp end region and 105 bp ofthe bpsD start region] and [ii] a bpsD-oxyD overlapping fragment [341 bp; contains 185 bp of the bpsD end region and 137 bp of the oxyD start region] [Fig . 2] . Thus, the existence of transcriptiontermination sites between bhp and bpsD as well as between bpsDand oxyD could be excluded, and therefore the three genes oxyD,bhp, and bpsD are part of one operon.

 
Inactivation of P450 monooxygenase gene oxyD. The fact that oxyD is cotranscribed with bhp and bpsD underscored the possibility of a coordinated function of these genes . To prove the participation of OxyD in ß-HT synthesis,we constructed a null mutant of A . balhimycina with an in-frame deletion within oxyD [OP090] . The gene replacement plasmid pOP2 [for construction details, see Materials and Methods], containing the oxyD gene with a 390-bp in-frame deletion, was used to transformthe A . balhimycina wild-type strain by means of a modified directtransformation method [21] . About 150 erythromycin-resistanttransformants were obtained, indicating an integration of pOP2via a first homologous recombination process . Nine randomlyselected resistant colonies were tested for the ability to producebalhimycin in a bioassay . One of these colonies [OP090v] lackedproduction of an active compound . In this case, the homologous fragment frOP3 most likely was used for the integration, resulting in a bhp-bpsD-oxyD operon with an oxyD gene with an in-framedeletion [Fig . 3] . Obviously, the intact oxyD gene copy downstreamwas inactive, most likely because of the missing natural promoter.

 
To obtain a deletion mutant, a second homologous recombination process was essential [Fig . 3] . To provoke a second recombination,we placed strain OP090v under stress conditions as describedpreviously [25], using temperature shifts and ultrasound treatment.After the application of the stress protocol, 500 colonies wereexamined on R5 plates with and without erythromycin . Four ofthe tested colonies lacked erythromycin resistance, indicatingthe loss of pOP2 . The balhimycin production abilities of thesecolonies were tested in a bioassay . One colony [OP266] was ableto produce balhimycin again, indicating a crossover event inthe same homologous region of pOP2 as that used for the integration.In contrast, the other three colonies [OP090, OP163, and OP364]were unable to produce an active compound, indicating an exchangeof the wild-type allele with the deleted oxyD gene as a resultof the second recombination event [Fig . 4A].

 
In the case of the mutant strain OP090, the in-frame deletionof oxyD was verified by PCR analysis, with total DNA used asa template [data not shown].

OP090 can be complemented by an additional oxyD gene copy. To demonstrate that the loss of balhimycin production in OP090was the result of only the deletion of oxyD and not of any additional mutational event, we introduced a complete copy of oxyD into the genome of OP090 by using the integrative vector pSET152. The integration of the complementation plasmid pSET-oxyD [for construction details, see Materials and Methods] into the OP090 chromosome resulted in the complemented mutant OP090k . PCR experiments with the total DNA of OP090k revealed that the integration of pSET-oxyD occurred at a neutral position in the genome, mostlikely at a C31 attachment site, and not via homologous recombinationinto the chromosomal oxyD locus [data not shown] . A bioassaywith the supernatant of OP090k demonstrated the restorationof balhimycin production [Fig . 4B].

This result confirmed that the failure of OP090 to produce balhimycin was a result of only the deletion of oxyD . Therefore, OxyD plays an essential role in the balhimycin biosynthesis process . In further LC-ESI-MS investigations, no intermediates or variantsof a higher molecular mass than 200 Da were detected in theculture filtrate of OP090 [data not shown], indicating the participationof OxyD in an early biosynthesis step.

OxyD participates in the ß-HT formation pathway. The previous data showed an essential function of OxyD in anearly reaction of balhimycin biosynthesis, most likely withinthe ß-HT formation pathway . To examine the involvementof OxyD in the synthesis of ß-HT, we performed cross-feedingexperiments [see Materials and Methods] with OP090, the bhpdeletion mutant OP696 [25], and the bpsD disruption mutant BpsD-cat [27] . In a first control experiment, the cross-feeding propertybetween OP090 and the null mutant JR1 [blocked in HPG and DPGsynthesis [23]] was investigated . Successful cross feeding wasdemonstrated by the appearance of an inhibition zone in bothcases [Fig . 5A] . The ability of OP090 to produce balhimycinin the neighborhood of JR1 demonstrated that OP090 had takenup an intermediate that was excreted by JR1 and converted itto balhimycin . This intermediate was likely a low-molecular-weightcompound, since A . balhimycina is not able to take up intermediateswith high molecular weights, such as, for example, the linearheptapeptide or the aglycone.

 
In further studies, the combinations OP090-OP696 and OP090-BpsD-cat were tested . No inhibition zones were detectable in the bioassays [Fig . 5B and C] . The lack of cross feeding in these cases demonstratedthat OP090 is blocked in the same pathway as OP696 and BpsD-cat,namely, ß-HT synthesis . To further prove the inhibitionwithin the ß-HT formation pathway, we incubated OP090in liquid medium containing ß-HT dissolved at a concentrationof 1 mg/ml . The harvested supernatant was then tested in a bioassay.An inhibition zone indicated the production of an active compoundby OP090 in the presence of ß-HT [Fig. 4C].

LC-ESI-MS studies confirmed that this active compound in the supernatant was balhimycin [Fig . 6] . The data from cross-feedingexperiments and ß-HT feeding experiments clearly demonstratethat OP090 is blocked in ß-HT synthesis.

 
Determination of chlorination time point. A previous analysis of bhaA deletion mutants demonstrated thechlorination activity of BhaA in balhimycin biosynthesis [25]. Attempts to establish an in vitro assay for the halogenatingenzyme BhaA were not successful, probably because the naturalsubstrate of BhaA is not available [K . H . van Pée, personalcommunication] . We therefore intended to define the time pointof chlorination [and thereby the substrate of BhaA] by usingdifferent mutants that were affected in balhimycin production.

Chlorination of balhimycin does not occur before or during formation of ß-HT. To exclude possible chlorination before or during ß-HT formation, we used the oxyD null mutant strain OP090 [see above], which is blocked in ß-HT synthesis, in feeding studies with nonchlorinated ß-HT . After 5 days of growth,the supernatant was harvested and investigated by LC-ESI-MSanalysis . The resulting mass spectrum unambiguously showed theproduction of balhimycin [molecular mass, 1,445 Da] [Fig . 6]and of variants that were also found in the culture filtrateof the wild-type strain [data not shown] . To confirm that chlorinated balhimycin was synthesized, we measured the isotopic patterns[Fig. 6] . They were identical to the theoretically calculated pattern of twofold chlorinated balhimycins.

These data clearly demonstrate that the halogenase BhaA is ableto chlorinate either ß-HT or an intermediate derivedfrom it . In the case of a tyrosine precursor as a natural substrateof BhaA, a conversion of ß-HT to chlorinated balhimycinshould not have taken place . Therefore, a chlorination reactionat a biosynthetic stage earlier than free ß-HT canbe excluded.

Chlorination of balhimycin does not occur with free ß-HT as a substrate of the halogenase. The data obtained from feeding studies with nonchlorinated ß-HTrevealed that the earliest time point of chlorination is therelease of ß-HT from BpsD . Therefore, we investigatedwhether the halogenase BhaA can use free ß-HT as asubstrate . In this case, CHT should represent a natural building block for heptapeptide backbone synthesis by the NRPS modules. To investigate whether CHT can be activated and introduced intothe peptide core by peptide synthetase modules two and six,we fed CHT to strain OP090 . Subsequently, we used a bioassayto analyze the biological activity of the supernatant after4, 10, 24, and 48 h and after 5 days of incubation . No activitywas detectable at any of the tested time points [Fig . 7] . Whereasthe supplementation of nonchlorinated ß-HT in a controlexperiment led to the biosynthesis of active balhimycin [Fig.7] after 24 h, the bioassay data revealed that the block ofß-HT synthesis in OP090 cannot be complemented byCHT . These data were independently confirmed by LC-ESI-MS studiesof culture filtrates.

 
One reason for this could have been the general inability ofA . balhimycina cells to take up the CHT dissolved in the medium.To exclude this possibility, we investigated the CHT concentrationof the isolated supernatant probes by HPLC . Time-dependent monitoringof culture filtrates with HPLC revealed a distinct decreasein the CHT concentration during the incubation time, comparableto the decrease in the ß-HT concentration in the controlexperiment [Fig. 8] . In contrast, the concentrations of bothamino acids were stable in cell-free medium, excluding spontaneous degradation as the reason for the reduction of dissolved CHTand ß-HT.

 
Thus, like ß-HT, CHT is certainly taken up, but itcannot be used as a substrate for peptide synthesis . Therefore,we can exclude the possibility that free chlorinated ß-HTis a naturally occurring precursor . This fact clearly pointsto a chlorination time point later than the stage of free ß-HT,most likely during the nonribosomal synthesis of the balhimycinheptapeptide core.

 
For our studies on the role of the putative P450 monooxygenaseOxyD, we constructed the oxyD in-frame deletion mutant A . balhimycina OP090 . Bioassays showed that OP090 lacked the ability to produce active balhimycin . The observed defect was restored by the integration of an intact oxyD gene . Additional mutations or polar effects on the genes downstream of oxyD could therefore be excluded as a putative cause of the OP090 phenotype . Thus, the production of active balhimycin by OP090 in the presence of ß-HT clearly identified OxyD as an enzyme that is, like Bhp and BpsD, essential for ß-HT synthesis . The functional cooperationof these genes is also reflected on the DNA level: the threegenes are part of one common operon, which guarantees coordinatedexpression.

The cotranscription of genes whose enzyme products form a functional unit can also be found in other biosynthetic gene clusters. cloQ and cloR in the clorobiocin cluster of Streptomyces roseochromogenesDS 12.976 as well as novQ and novR in the novobiocin clusterof Streptomyces spheroides are likely to form an operon . Theseenzymes are involved in the biosynthesis of the prenylated 4-hydroxybenzoatemoiety [RingA] derived from tyrosine [24] . Furthermore, polyene antibiotic gene clusters contain very large genes encoding polyketide synthases, some of which seem to be cotranscribed . Such transcripts could be extremely long, e.g., encompassing >47 kb synthesizedfrom the nysA [encodes the ketosynthase-acyl transferase-dehydratase-acyl carrier protein], nysB [encodes a bimodular protein which catalyzes the first two cycles of chain extension], and nysC [encodes extension modules 3 to 8, organized into hexamodular proteins] genes in Streptomyces noursei or from the corresponding amph genes in Streptomyces nodosus, the producers of nystatin and amphotericin, respectively [2] . However, the coexpression ofgenes forming a functional unit is not a general observation.For example, expression studies of biosynthetic genes of themacrolide antibiotic tylosin revealed no coexpression and even no coregulation of the five genes coding for the polyketide synthases [30].

OxyD resembles OxyA, OxyB, and OxyC, which participate in linkage of the aromatic residues [6], but it has more significant similarityto the P450 monooxygenases NovI [56% similarity; 42% identity]and NikQ [52% similarity; 34% identity] . NovI is responsiblefor the ß-hydroxylation of a tyrosine intermediate covalently bound to a NRPS protein in the biosynthesis of the aminocoumarin antibiotic novobiocin [9] . NikQ is a hydroxylatingenzyme in the synthesis of ß-hydroxyhistidine as aprecursor of nikkomycin antibiotics [10].

In accordance with these reactions, we propose a hydroxylating function for OxyD, with a tyrosine bound to the NRPS BpsD asa substrate . This speculation is confirmed by the presence ofthe tyrosine-specific adenylation domain in BpsD [9] . The inactivationof OxyD in A . balhimycina OP090 prevented the formation of ß-HT,which is an important building block of the balhimycin heptapeptidebackbone . The oxyD mutant, like mutants defective in bpsD andbhp, is an in-frame null mutant and does not produce balhimycinprecursors since no active compound was detectable in the bioassayand no higher-molecular-weight balhimycin variant could be identifiedby the LC-ESI-MS analysis of the OP090 supernatant . Obviously,no naturally occurring alternative amino acid of ß-HT,for example, tyrosine, can be incorporated at positions twoand six of the heptapeptide backbone . This is in accordancewith the results of tyrosine feeding studies with the bhp-deficientstrain A . balhimycina OP696 [35].

In further studies, we used the oxyD mutant strain OP090 as a suitable tool for analyzing the chlorination time point in balhimycin biosynthesis . Even though BhaA, which belongs tothe group of NADH/FAD-dependent halogenases, was identifiedas the enzyme catalyzing the chlorination of balhimycin at bothpositions [25], the substrate of this reaction has not beenidentified yet . The first hints that chlorination is not a tailoringreaction at a very late stage of glycopeptide biosynthesis wereobtained with the mutant A . balhimycina SP1-1, which accumulatedfully chlorinated linear heptapeptides as natural intermediates[22, 31] . This means that the chlorination reaction occurs at a time point prior to oxidative cyclization through oxygenases OxyA/B and -C and subsequent glycosylation-methylation reactions. Since many identified NADH/FAD-dependent halogenases probablyuse substrates of a low molecular weight, for example, phenolsand pyrrols [33], one might have speculated that chlorination in balhimycin biosynthesis occurs with tyrosine or ß-HT as the natural substrate of BhaA . However, the incorporationand subsequent conversion of fed ß-HT as well as thefailure of the NRPSs BpsA and BpsB to use CHT as a buildingblock for heptapeptide synthesis clearly exclude this possibility.Heptapeptide synthesis itself and further modification reactionssuch as epimerization are catalyzed by the NRPSs BpsA, BpsB,and BpsC [26] according to well-known rules [for reviews, seereferences 17 and 29].

The data obtained in this study are the first evidence thatthe chlorination process must be a reaction during heptapeptide synthesis, similar to those normally catalyzed by domains incorporated in the NRPS . Therefore, a close association of the halogenase with the NRPS machinery must be postulated.

 
This work was supported by grants from the European Union [MEGATOP, QLK3-1999-00650; and COMBIG-TOP, LSHG-CT-2003-503491] and the Deutsche Forschungsgemeinschaft [DFG] [Wo485/3-3 and SU 239/3-3].The work of R . D . Süssmuth was supported by an Emmy-Noether-Fellowship for young investigators of the DFG [SU 239/2-1].

We thank E . Takano for a critical reading of the manuscript.

 
* Corresponding author . Mailing address: Mikrobiologie/Biotechnologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany . Phone: 49 7071 2976944 . Fax: 49 7071 295979 . E-mail: wolfgang.wohlleben@biotech.uni-tuebingen.de .

Present address: GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Entwicklungsgenetik, D-85764 Neuherberg, Germany.

Present address: Boehringer Ingelheim Pharma GmbH & Co.KG, 88397 Biberach, Germany.

Present address: Combinature Biopharm AG, D-13125 Berlin, Germany.

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