Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster

Abstract Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one—vemR—that encodes a transcriptional activator of the large ATP‐binding LuxR‐like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co‐expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin‐producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.


Introduction
Polyketides are structurally diverse natural products from plants and microorganisms that exhibit ab road range of biological activities. [1][2][3] They are assembled through aprocess similar to fattya cid biosynthesis, involving one or more rounds of decarboxylative condensation of an (alkyl) malonyl thioester extender unit with an acyl thioester starter unit. Several distinct classes of polyketide synthases exist;t hey are differentiated by their architectures and catalytic mechanisms. [4] The type IP KSs each consist of one or more multifunctional proteins containing various catalytic domains that are used to assemblet heir metabolic products.W ith the exception of PKSs that utilise trans-acting acyl transferases, [5] at ype IP KS generally contains am inimum of three domains: ak etosynthase (KS), an acyl transferase (AT) and an acyl carrier protein (ACP). Optional ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) domainsd efine the degree of b-carbonp rocessing after each round of chain elongation. [6] Type IP KSsc an be divided into two subgroups.
The first subgroup is termed "modular"; at ypicalr epresentative consistso faseries of modules, each of which contains as et of distinct catalytic domainst hat are responsible for one round of chain elongation and subsequent b-carbonm odification in the overall chain assembly process. The archetypal example of such PKSs is 6-deoxyerythronolide Bs ynthase (DEBS), which is involved in the biosynthesis of the antibiotic erythromycin in the actinobacterium Saccharopolyspora erythraea. [7] The second subgroup is termed" iterative" and is exemplified by 6-methylsalicylic acid synthase (MSAS), found in many fungi.
Type II PKSs each consist of as eries of monofunctional enzymest hat are used iteratively in the assembly of polycyclic aromatic metabolites (e.g.,a ctinorhodin and tetracycline) and are mainly found in Actinobacteria. [8][9] Comparative transcriptional profiling of a DbldM mutant of Streptomycesv enezuelae with its unmodified progenitor revealed that the expression of ac ryptic biosynthetic gene cluster containing both type Ia nd type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encodea nu nusualb iaryl metabolite, which we named venemycin, and potentially halogenatedd erivatives, contains 16 genes including one-vemR-thate ncodes at ranscriptional activatoro ft he large ATP-binding LuxRlike (LAL) family.C onstitutive expression of vemR in the DbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure.
Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologoush ost Streptomyces coelicolor also resulted in venemycin production.A lthough the gene cluster encodes two halogenases and af lavin reductase, constitutive expression of all three genes led to the accumulation only of am onohalogenatedv enemycin derivative, both in the native producer and the heterologous host. Ac ompetition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resultedi nt he preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.
Type III PKSs (e.g.,c halcone and stilbene synthases) were first identified in plants, but have since been found in many eubacteria. [10] They are multifunctional enzymes that catalyse elongation of diversea cyl-CoAs tarter units with one or more malonyl-CoA extender units to form poly-b-ketoacyl-CoA intermediates that undergo ar ange of cyclisation reactions to form diversea romatic products. [4,7,11] This type of PKS is exemplified by RppA,w hich assembles 1,3,6,8-tetrahydroxynaphthalene (THN) in several Streptomyces species. [12][13][14][15] Streptomyces is the largestg enus within the Actinobacteria phylum. Streptomyces species are aprolific sourceo fantibiotics and other bioactive natural products with ab road range of applicationsi nb oth medicine and agriculture. [16] The recent sequencing of alarge number of actinobacterial genomes has revealed the presence of numerous gene clusters with the potential to direct the biosynthesis of new specialised metabolites. However,i nm ost cases the metabolic productso fs uch cryptic gene clusters remaint ob ei dentified, presumably because they are not expressed under laboratory growth conditions. [17] Although wellk nown as ap roducer of chloramphenicol, Streptomyces venezuelae ATCC 10712 failed to produce this antibiotic in our laboratory under ar ange of culture conditions. However,f or reasons that we do not yet understand, deletion of bldM,w hiche ncodes an atypical response regulator required for morphological development, [18] activated transcriptiono ft he chloramphenicol biosynthetic gene cluster. [19] Here we report the discovery of an unusualb iaryl polyketide as the metabolic product of ac ryptic polyketide biosynthetic gene cluster that was identifiedb yc omparative transcriptional analyses of the wild-type and DbldM strains of S. venezuelae.

Results and Discussion
Identification of acrypticpolyketide biosynthetic gene clusterinS. venezuelae and prediction of its biosynthetic product Comparative microarray analysiso fw ild-type S. venezuelae and ac ongenic bldM mutant (strain SV13) [18] revealed the presence of ap reviously unidentified cryptic polyketide biosynthetic gene cluster( vem), expression of which was markedly upregulated in the mutant (Figure 1). The cluster is approximately 29.5 kb in size and contains 15 upregulatedg enes: sven0482, sven0483,a nd sven0485-sven0497.T he transcriptionalp rofiles of the flanking genes sven0477-sven0481 and sven0498-sven0502,w hich weree xpressed at low levels both in the wildtype strain and in the bldM mutant (data not shown), indicated that they were unlikelyt ob ef unctionally relatedt ot he vem cluster (sven0482-sven0497). From the amino acids equence similarity of the encoded proteins to those present in the NCBI protein database we predicted their putative functions (Table 1) and proposed ab iosynthetic pathway (Scheme1) that, if correct, would result in the productiono fanew biaryl metabolite-which we named venemycin-and halogenated derivatives.
We suggest that 3,5-dihydroxybenzoic acid serves as astarter unit for the assemblyo fv enemycin by the type Im odular PKSs VemG and VemH. Conserved domain searches revealed that VemG contains at its Nterminus al oading module consisting of an adenylation (A) domain, ak etoreductase( KR) domain (predicted to be nonfunctional because it lacks ac anonical YXXXNm otif at its active site, even though it appears to contain an NADPH binding site) and an ACP domain. The Adomain has 63 %a mino acid sequence similarity to the correspondingd omain at the Nterminus of the type Im odularP KS responsible for the assembly of kendomycin, which has been proposed to load a3 ,5-dihydroxybenzoic acid starteru nit onto the PKS. [20] At its Cterminus, VemG has ac hain elongation module that containsK S, AT and ACP domains. VemH is predicted to contain as econd chain elongation module consisting of KS, AT and ACP domains, followed by at hioesterase (TE) domain.T he AT domains in VemG andV emH are both predict- ed to be specific for malonyl-CoA. [27] The TE domain likely catalyses releaseo ft he fully assembled polyketide chain to form the pyrone, although hydrolytic releaset of orm as omewhat less stable 3,5-diketo acid would also be possible. VemI is ap hosphopantetheinylt ransferase (PPTase) that is proposed to be responsible for posttranslational phosphopantetheinylation of the three ACP domains within the modular PKS;s urprisingly, because it is embedded in the cluster, transcription of vemI (sven0484)w as not markedly increased in the bldM mutant ( Figure 1), and it is conceivable that under the growth conditions used this function is mainly performed by one of the other PPTases encoded in the S. venezuelae genome by sven0914, sven6190, sven6269 and sven4419 (the last of these is likely to be the PPTase used in fatty acid synthesis). Either of the aromatic rings of venemycin could then be halogenated by one or both of the two putative flavin-dependent halogenases, VemJ and VemK, with the FADH 2 cofactor required by these enzymes being supplied by the flavin reductase VemL. VemN andV emO belong to the major facilitator superfamily (MFS) transporters and are presumably involved in the export of venemycin. VemR is am ember of the large ATP-binding LuxR-like (LAL) family of transcriptional regulators and is proposed to function as ap athway-specific activatoro ft he biosynthetic gene cluster.T he protein contains aN -terminal AAA-ATPase domaina nd aC -terminal LuxR family DNA-binding domain with ah elix-turn-helix motif. LAL homologues have been shown to activate the production of severala ctinomycete specialised metabolites, including pikromycin (PikD), [28] rapamycin (RapH) [29] and the stambomycins (SAMR0484). [30] Sven0490i sh omologous to isochorismatases that convert isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate. These enzymes commonly participate in the biosynthesis of nonriboso-mal peptide siderophores, and there is no apparent role for Sven0490invenemycin biosynthesis.

Identification of venemycin and ac hlorinated derivative
Our proposed biosynthetic pathway (Scheme 1) predicted that S. venezuelae should produce ap otentially new metabolite with the molecular formula C 11 H 8 O 5 ,a sw ell as halogenated derivatives. On the basis of this prediction, we set out to identify and characterise the productso ft he vem gene clusterb yc omparativem etabolite profiling of culture supernatants from the S. venezuelae DbldM mutant (strain SV13) and its parental strain after growth for four days in liquid maltose-yeast extract-malt extract medium made with tap water (MYM-TAP) and supplemented with trace elements. LC-HRMS identified molecular ions corresponding to [M+ +H] + for venemycin [m/z calcd for C 11 H 9 O 5 + :2 21.0444;f ound:2 21.0443( an absorbance maximum at 324 nm waso bserved in the UV/visible spectrum for this species;F igure S1 in the SupportingI nformation)] and am onochlorinated derivative [m/z calcd for C 11 H 8 O 5 Cl + : 255.0055; found:2 55.0051 ( Figure S2)] that were present only in the culture supernatant of the mutants train. The compounds were detectable at 48 ho fg rowth and their levels peakedb etween 72 and 96 h; after 120 ht hey had declined by 30 %( data not shown).
Heterologous expression of the vem gene clusterinS. coelicolor and structure elucidation of venemycin Because the levels of production of venemycin and its monochlorinated derivative by the S. venezuelae DbldM mutant were insufficient to permit furthers tructurala nalysis, and to confirm that the vem gene cluster does indeedd irect venemycin biosynthesis, we set out to express the gene cluster in ah eterologous host. The cosmid derivative pIJ13035 (Table S1) containing the entire vem gene clusterw as introduced into Streptomyces coelicolor M1152 [31] and M1316 [32] (a derivativeo fM 1152 from whicht wo native type III PKS genes have been deleted; Ta ble S1)b yc onjugation from Escherichia coli.C hromosomal integration was confirmedbyPCR analysis, yielding S. coelicolor M1818 and M1819, respectively.T he strains were grown as described above,a nd their culture supernatants were analysed by LC-MS. Neitherv enemycin nor its chlorinatedd erivativew as detected (data not shown);this probably reflects alack of transcriptionoft he vem gene clusterint he heterologoushosts.
To surmount this problem, vemR,e ncoding ap utative transcriptional activatoro ft he LAL family,w as clonedd ownstream of the constitutive ermE*p romoter within the integrative pIJ10257v ector, [33] and the resulting plasmidp IJ13028 was introduced into both S. coelicolorM 1818 and M1819 by conjugation from E. coli,y ielding S. coelicolor M1822( M1152 + vem cluster + ermE*p::vemR)a nd S. coelicolor M1825( M1316 + vem cluster + ermE*p::vemR), respectively.C hromosomal integration of pIJ13028 in each of the recipient strains was confirmed by PCR analysis (data not shown). Cultivation of both M1822a nd M1825 resultedi nt he productiono fam etabolite with the same molecular formula, retentiont ime and MS/MSfragmentation pattern as venemycin produced by S. venezuelae SV13 ( Figure 2). Sometimes, butn ot always, av ery small amount of as ingly chlorinated derivativeo fv enemycin was also detected in the culture supernatants of these strains( data not shown). S. coelicolor derivatives containing pIJ13028 but lacking the vem gene cluster did not produce venemycin or its chlorinated derivative( data not shown). Thisc onfirmed that pIJ13035 contains all of the genes required for venemycin biosynthesis. Introduction of pIJ13028 into wild-type S. venezuelae (yielding M1815)a lso resulted in venemycin production (Figure 2), thus providing furthere videncet hat VemR is at ranscriptional activator.L evels of production were further increased when the same plasmid was introduced into the S. venezuelae DbldM mutant( to give M1817), yieldings ufficientv enemycin for structure elucidation by NMR spectroscopic analysis( Scheme 2 and Ta ble S3). Venemycin was purified from M1817bysemipre-parativeH PLC and analysed by 1 Ha nd HSQC andH MBC NMR experiments (Figures 3a nd S3-S6). The NMR analyses established that 3,5-dihydroxybenzoic acid had co-purified with venemycin, consistent with its proposedr ole as an intermediate in venemycin biosynthesis (Figures S3-S7). As earch of the Sci-Finder database established that although venemycin had not been reported previously as an atural product it is ak nown synthetic compound with potent superoxide anion radical scavenging activities. [34]

Halogenationo fv enemycin
Because production levels of the monochlorinated derivative of venemycin werelow and no doubly halogenated venemycin derivatives could be detected despite the presence of two genes (vemJ and vemK)e ncoding putative FADH 2 -dependent halogenases, we decided to express vemJ, vemK and vemL (encoding af lavin reductase) from the strong constitutive ermE* promoter.T he genes were cloned into the conjugative multicopy vector pIJ12477 [35] (Table S1), and the resulting plasmid-pIJ13029( ermE*p::vemJKL)-was introduced into S. venezuelae M1815 (wild-type + ermE*p::vemR)a nd M1817 (DbldM + ermE*p::vemR), as well as S. coelicolor M1822 (M1152 + vem cluster + ermE*p::vemR)a nd M1825 (M1316 + vem cluster + ermE*p::vemR), yielding strains M1827, M1831, M1835 and M1839, respectively.T he strains were grown as before in liquid MYM-TAP mediums upplemented with trace elements, and the culture supernatantsw ere analysed by LC-HRMS in negativeion mode, which resultedi ni ncreased sensitivity relative to the earlier analyses carried out in positive-ion mode.V enemycin and its monochlorinated derivative were detected in all of the cultures (data not shown), but the amounts produced were still insufficient to permit further structural characterisation of the chlorinated form, and no polyhalogenated venemycin derivatives were detected. From HPLC profiles with UV-a nd visible-range detection, the levelso fp roduction of venemycin and the monochlorinated form in S. coelicolor strains M1835 and M1839 were generally about half those observed in the S. venezuelae DbldM derivative M1817( data not shown). The two heterologous hosts produced similar amounts.
Some flavin-dependent halogenases prefer bromide over chloride, [36] but because chloride is the most abundant halide in commonly used culture media, it is chlorine rather than bromine that is usually incorporated into metabolicp roducts under laboratory conditions. To test whether bromide ions are accepted or even preferred by VemJ/VemK, S. venezuelae M1831( DbldM + ermE*p::vemR + ermE*p::vemJKL)a nd S. coelicolor M1835 (M1152 + vem-cluster + ermE*p::vemR + ermE*p::vemJKL)w ere cultivated in am inimal medium( MM) supplemented with NaCl or NaBr.Amonobrominated venemycin derivative was readily detected in the culture supernatants of both strains ( Figure S8). Because heterologouse xpression in S. coelicolor had workedw ell (Figures 3a nd S8), we repeated the experiment with M1822a nd M1835i nm odified R3 mediums upplemented with NaCl, NaBr,o re quimolar amounts of both. A1 3:1r atio of monobrominated to monochlorinated venemycin derivatives was observed in the culturec ontaining equimolar amounts of NaCl and NaBr,t hus indicating that bromide is the preferred substrate for one or both of the halogenases encoded in the venemycin biosynthetic gene cluster ( Figure 3). Although the monobrominated venemycin derivative constituted almost6 0% of the venemycin-related metabolites produced in the culture containing NaBr,t he amountp roduced was not sufficient to permitfurtherstructural analysis.
Halogenation of an atural product can significantly affect its biological activity,i ncluding its antibacterial activity. [37][38][39] We thus investigated the activity of culture supernatants of M1835 containing venemycin, and its monochlorinated and monobrominatedd erivatives,a gainst Micrococcus luteus and E. coli,b ut no growth inhibition was observed (data not shown).

Transcriptionalorganisation of the vem gene cluster
Inspection of the sequence of the vem gene cluster (Figure 1) revealed the presence of multiple transcription units, and several potentialo perons. To throw light on how expression of the gene clusterm ight be regulated, and to provide ab asis for future attempts to increase the level of productivity-by engineering enhanced levelso ft ranscription,f or example-the operon structure of the vem gene cluster was determined. RNA was isolatedf rom mycelium of S. coelicolor M1825 (M1316 + vem cluster + ermE*p::vemR)a fter two days of incubation, corresponding to the onset of venemycin production, and cDNA was synthesised by at wo-step RT-PCR protocol with random hexamers in the presence of reverset ranscriptase (RT). The resultingc DNA was used as at emplate for PCR amplification with primers flanking 14 intergenic regions within the pro-  (Table S2). The fidelity of the primers used was confirmed by using pIJ13035 DNA as template. Each pair produced as ingleP CR product of the predicted size. The results suggested the existence of eight transcription units: vemN, vemD, vemI, vemGH, vemJK, vemL-sven0490-vemO, vemR and vemABCEF (Figure 4). Attempts to use RT-PCR to amplify cDNA derived from hrdB,e ncoding the housekeeping s factor of S. coelicolor,b yu sing RNA treated with and without reverse transcriptase and 35 cycles of amplification confirmed the absence of genomic DNA.

Conclusion
By combining microarray analysiso faS. venezuelae DbldM mutant with bioinformatics analysis of its genome sequence, we were able to identify ab iosynthetic gene cluster encoding an unusualc ombination of modulart ype Ia nd type III PKSs that was predicted to assemble an ew biaryl natural productwhich we named venemycin-along with halogenated derivatives. Comparative analysis of the metabolites produced by the wild-type strain and the DbldM mutant by using UHPLC-ESI-Q-TOF-MS identified two compounds with molecular formulae corresponding to venemycin and am onochlorinated derivative. However,t he quantities of these metabolites were insufficient to permits tructure elucidation by NMR spectroscopic analysis. Thus, vemR,w hich encodes ap utative LAL transcriptional activator,w as constitutively expressed in S.venezuelae and in S. coelicolor derivativesc ontaining the cloned vem gene cluster.T his provided sufficient quantities of venemycin for NMR analysis,t hus confirming that it has the structurep redicted by bioinformatics. Although the vem gene cluster contains two halogenase genes (vemJ and vemK), we were only able to detect the production of monohalogenated venemycin derivatives. It is conceivable that one of the halogenases is not func-tional, or that each of the halogenases modifies venemycin at ad ifferent position and the monohalogenated venemycin derivatives are not substrates for the remaining halogenase. In ac ompetitive feeding experiment employing equimolar concentrationso fc hloride and bromide, the monobrominated venemycin derivative(s)w as/werep roduced at a1 3-fold higher level than the monochlorinated derivative(s), thus indicating that one or both of the halogenases has as ignificant preference for the larger halide ion. Although venemycin is produced in part by at ype III PKS, there weren os ignificant differences in the levels of production in M1152 and M1316, ad erivative of M1152 in which two type III PKSg enes have been deleted and in which precursor supply for heterologously synthesised venemycin might have been better.I tr emains to be seen whether this is true for other metabolites derived from malonyl-CoA. Our biosynthetic scheme has no role for sven0490 (encoding ap utative isochorismatase), and indeed, deletion of this gene in S. coelicolor M1825 had no effect on venemycin production (A. Thanapipatsiri, unpublished data). This is particularly surprising because sven0490 is co-transcribed with vemL and vemO.
Microarray analysis:R NA isolation from S. venezuelae and subsequent DNA microarray analysis were reported previously, [46] and the data were deposited in the arrayExpress database (https:// www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-2716. Briefly,d ata in CEL files produced by the chip scanner were normalised by using robust multi-array average (RMA) as implemented in the Affy package of Bioconductor (https://www.bioconductor.org/). Replicates were combined by using the lmFit and eBayes functions of the limma package of Bioconductor.T his resulted in at able of log 2e xpression values for each gene at various time points in the different strains. For the analysis reported here, the log 2e xpression values for the relevant genes (sven0477-sven0481, sven0482-sven0497 and sven0498-sven0502)i nt he bldM mutant and the wild-type strains were extracted from the genome-wide data set, centred to am ean of zero for each gene (by using the scale function of R) and then plotted by using R (https://www.R-project.org/). DNA and RNA manipulations:D NA extraction from E. coli was carried out by standard methods [42] or with aQ IAprep Spin Miniprep Kit (Qiagen). Restriction enzymes (Roche and NEB) were used according to the instructions provided by the manufacturers. PCR amplifications were performed with Ta qD NA polymerase (Qiagen). The amplification of DNA fragments used in the expression constructs was performed with Phusion High-Fidelity DNA Polymerase (NEB). Extraction of RNA from Streptomyces strains was performed with aR Neasy mini kit (Qiagen, Supporting Information). RT-PCR was performed with aM axima First Strand cDNA Synthesis kit (Thermo Scientific). Amplification of DNA from synthesised cDNA was carried out with Ta qDNA polymerase (Qiagen).
Annotation of the vem cluster:T he Streptomyces genome server (StrepDB:h ttp://strepdb.streptomyces.org.uk) was used to derive the amino acid sequences for each of the proteins encoded by the vem cluster.T hese were then used in BlastP searches of the NCBI protein database to assign the probable function of each gene.
Generation of at ransmissible cosmid for heterologous expression of the vem cluster in S. coelicolor:P CR targeting [41] of cosmid SV3E02, which contains the vem cluster,w as carried out to replace neo of the SuperCos1 vector with a5 247 bp oriT-attP-int-aac(3)IV fragment (flanked by SspI recognition sites), which was isolated by SspI restriction digestion of pMJCOS1 [47] (now known as pIJ10702, Figure S9). Replacement occurred through double-crossover recombination at homologous flanking sequences to yield the conjugative and integrative pIJ13035, which was then introduced into the heterologous hosts S. coelicolor M1152 and S. coelicolor M1316 (Table S1) by intergeneric conjugation, [44] whereupon it integrated into the host's genome at the chromosomal fC31 attB site to yield S. coelicolor M1818 and S. coelicolor M1819, respectively.T he exconjugants were selected on SFM agar containing apramycin and nalidixic acid and streaked for ac ouple of passages on the same agar to ensure complete removal of the E. coli donor strain. Chromosomal integration of pIJ13035 was confirmed by PCR amplification of selected vem genes by using two primer pairs: SVEN0487TF/SVEN0487TR (flanking vemJ)a nd SVEN0493TF/ SVEN0493TR (flanking vemA,T able S2). The resulting S. coelicolor strains were grown until fully sporulated, and the spores were harvested and stored at À20 8Ci ng lycerol (20 %). [44] Constitutive expression of vemR:T he vemR coding sequence (from start to stop codon) was amplified from cosmid SV3E02 by using primers SVEN0492NdeIF1 and SVEN0492HindIIIR (Table S2) and blunt-end cloned into pBlueScript II KS(+ +)a tt he SmaI site. Restriction enzyme digestion and DNA sequencing by using M13 universal primers and designed sequencing primers SVEN0492NW1, SVEN0492NW2, SVEN0492W3, SVEN0492W4 and SVEN0492NW5 (Table S2) were used to verify the cloned fragment, which was then inserted downstream of the constitutive ermE* promoter in pIJ10257 (which integrates at the host's chromosomal fBT attB site [33] ), creating pIJ13028 in E. coli DH5a ( Figure S10). [42] pIJ13028 and pIJ10257 were then introduced by conjugation into S. venezuelae wild type, S. venezuelae SV13 (DbldM), S. coelicolor M1818 (M1152::pIJ13035) and S. coelicolor M1819 (M1316::pIJ13035). pIJ13028 was also introduced into S. coelicolor M1152 and S. coelicolor M1316 to generate vemR expression control strains. S. coelicolor ex-conjugants were selected on SFM agar containing hygromycin and nalidixic acid whereas MYM-TAP agar was used for the S. venezuelae strains. The resulting strains were streaked on the same media containing hygromycin and nalidixic acid until the strains were free of E. coli cells. The resulting recombinant strains were verified by PCR with primers pIJ86F1 and SVEN0492HindIIIR (Table S2) and grown until fully sporulated, and the spores were harvested and stored at À20 8Ci nglycerol (20 %).
Constitutive expression of halogenase genes:APCR fragment containing the coding sequences of vemJKL and the ribosomal binding site of vemJ was amplified by using primers SVEN0487-BamHIF and SVEN0489HindIIR and cloned into pBluescript II KS(+ +) cleaved with SmaI. The cloned fragment was verified by restriction enzyme digestion and DNA sequencing with use of M13 universal primers and designed sequencing primers SVEN0487W1, SVEN0488W1, SVEN0488W2 and SVEN0489W1 (Table S2) and inserted downstream of the constitutive ermE*p romoter in the multicopy pIJ12477 cut with BamHI and HindIII, creating pIJ13029 (ermE*p::vemJKL,F igure S10). pIJ13029 and pIJ12477 (vector-only control) were then introduced into Streptomyces strains by intergeneric conjugation as previously described. [44] S. coelicolor ex-conjugants were selected on SFM agar containing kanamycin and nalidixic acid, whereas MYM-TAP agar containing trace element solution and kanamycin was used for the S. venezuelae ex-conjugants. Ex-conjugants were verified by PCR amplification with primer pair pIJ86F1 and pIJ86R2 (Table S2), which anneal at sites within pIJ12477 220 and 229 bp from the BamHI and HindIII cloning sites of the vector,r espectively.T he resulting strains were grown on agar,a nd spores were stored as previously described.
Analysis of culture supernatants:S tationary-phase cultures were centrifuged at 13 000 rpm in af ixed angle rotor refrigerated microcentrifuge at 4 8Cf or 20 min to remove mycelium. Samples were analysed by using two different systems and sets of conditions: A) AS himadzu LC-MS system equipped with aN exeraX2 liquid chromatograph (LC30AD) fitted with aP rominence photodiode array detector (SPD-M20A) and an LC-MS-IT-ToF mass spectrometer:S amples (typically 5 mL) were injected onto aK inetex XB C 18 (2.6 mm, 100 , 50 mm by 2.1 mm) column (part no. 00B-4496-AN, Phenomenex, USA) fitted with aK rudKatcher Ultra HPLC inline filter (part no. AF0-8497, Phenomenex), kept at 40 8Ca nd eluted at af low rate of 0.6 mL min À1 with ag radient of 0.1 %f ormic acid in water and methanol, from 2% to 100 %m ethanol over 7min. Data acquisition and analysis were performed with LCMS solution version 3 (Shimadzu). B) AD ionex 3000RS UHPLC coupled to aB ruker MaXis Q-TOF mass spectrometer with an Agilent Zorbax Eclipse plus column (C18, 100 2.1 mm, 1.8 mm):M obile phases consisted of A( water with 0.1 % formic acid) and B( acetonitrile with 0.1 %f ormic acid). Ag radient of 5% Bt o1 00 %Bin 15 min was employed with af low rate of 0.2 mL min À1 ,with UV absorption set at 254 nm.
Assay of antibacterial activity:C olonies of M. luteus ATCC4698 and E. coli ATCC25922 freshly grown on lysogeny broth (LB) agar plates were used separately to inoculate LB broth (10 mL) followed by overnight cultivation at 30 8C. Overnight culture (0.2 mL) was used to inoculate LB (10 mL) and cultivation at 30 8Cw as continued until an OD 600 of 0.6-0.8 was reached. The culture (1 mL) was added to LB-agar medium (50 mL) at about 45 8C, and 25 mL were poured into 10 10 cm plates. Culture supernatant (100 mL) was added to wells made in the solidified agar,a nd plates were incubated at 30 8Cf or 24-48 h, after which they were monitored for the appearance of inhibitory halos. Kanamycin (25 mgmL À1 )w as used as positive control.