Accepted Article Title: Genome mining of Streptomyces sp. Tu¨ 6176: characterization of nataxazole biosynthesis pathway

Authors: Carlos Olano; Carolina Cano-Prieto; Raul ´ Garc´ıa-Salcedo; Marina ´ ˜ Hans-Peter Fiedler; Carmen Mendez; ´ Sanchez-Hidalgo; Alfredo F. Brana; Jose´ A. Salas

This manuscript has been accepted after peer review and the authors have elected to post their Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article.

To be cited as: ChemBioChem 10.1002/cbic.201500153 Link to VoR: http://dx.doi.org/10.1002/cbic.201500153

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ChemBioChem

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Genome mining of Streptomyces sp. Tü 6176: characterization of nataxazole biosynthesis pathway

Carolina Cano-Prieto[a], Raúl García-Salcedo[a], Marina Sánchez-Hidalgo[a], Alfredo F. Braña[a], Hans-Peter Fiedler[b], Carmen Méndez[a], José A. Salas[a] , Carlos Olano*[a]

[a] C. Cano-Prieto, Dr. R. García-Salcedo, Dr. M. Sánchez-Hidalgo, Prof. A. F. Braña, Prof. C. Méndez, Prof. J. A. Salas, Dr. C. Olano. Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), Universidad de Oviedo, 33006 Oviedo, Spain E-mail: [email protected] [b] Prof. H. P. Fiedler Mikrobiologisches Institut, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany

Abstract Streptomyces sp. Tü 6176 produces cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of thirty eight putative secondary metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, antibiotic hygromycin B and

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ionophores enterobactin and coelibactin. Nataxazole biosynthesis gene cluster has been identified and characterized showing the absence of an O-methyltransferase coding gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism since AJI9561 shows antibiotic activity while nataxazole is inactive. Moreover, expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the heterologous production of AJI9561. Nataxazole biosynthesis requires shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to 4-fold by deregulating the gene cluster expression. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5hydroxynataxazole derives from nataxazole by the activity of a yet unidentified oxygenase, which might imply a cross-talk between the nataxazole biosynthesis pathway and a still unknown pathway.

Keywords: AJI9561, Benzoxazole biosynthesis, Genome mining, Iterative type I PKS

Introduction Streptomyces sp. Tü 6176 is the producer of cytotoxic benzoxazole nataxazole.[1] The benzoxazole family of compounds includes other natural products with cytotoxic activity such as UK-1[2] and AJI9561[3] produced by Streptomyces spp. (Scheme 1). Furthermore benzoxazole antibiotics calcimycin,[4] A33853[5] and caboxamycin

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(Scheme 1)[6] are also produced by Streptomyces spp. The characteristic benzoxazole moiety is a heterocyclic structure composed by a benzene-fused oxazole ring forming a benzoxazole motif. Caboxamycin, calcimycin and A33853 have only one benzoxazole motif while nataxazole, UK-1 and AJI9561 have two benzoxazole motifs. Nataxazole (NAT) shows cytotoxic activity against several human tumor cell lines. NAT is more active against AGS (gastric carcinoma) human tumor cell line than UK-1 and equivalent to UK-1 against MCF7 (breast cancer) and HepG2 (liver hepatocellular carcinoma) cell lines.[1] Based on the cytotoxic activity mechanism of UK-1[7] and considering that NAT and UK-1 produce an accumulation of AGS cells in S phase, it has been proposed that NAT is an inhibitor of topoisomerase II binding to double-stranded DNA in a metal ion-dependent fashion.[1] Due to their broad range of biological activities, benzoxazoles constitute an important class of therapeutic and biotechnological compounds, drawing the attention of chemists in order to synthesize novel derivatives.[8] However, biosynthesis of benzoxazole family of compounds has not been explored in detail, being calcimycin biosynthesis pathway the only one reported. Calcimycin is the most complex natural benzoxazole since it is composed by three moieties: a spiroketal ring synthesized by a type I polyketide synthase (PKS), an α-ketopyrrole derived from L-proline and modified by a non-ribosomal peptide synthetase (NRPS), and a benzoxazole unit whose precursor is 3-hydroxyanthranilic acid (3-HAA).[9] We were interested in the biosynthesis of NAT to increase the knowledge on how different benzoxazole compounds can be synthesized by actinomycetes. Structurally, NAT is formed by three moieties, two correspond to 3-HAA while the third is 6-methylsalicylic acid (6-MSA) (Scheme 1). Different biosynthesis pathways have been reported for the biosynthesis of 3-HAA. In phenazine pathway, 3-HAA

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derives from chorismate by the action of an isochorismatase and an anthranilate synthase.[10] Same origin has been reported for benzoxazole calcimycin.[9] On the other side, in pyrrolobenzodiazepine sibiromycin biosynthesis pathway, 3-HAA derives from L-tryptophan that is modified via the kynurenine pathway through the activity of a kynurenine 3-monoxygenase, an aryl formamidase, a tryptophan-2,3-dioxygenase and a kynurenine hydrolase.[11] On the other hand, 6-MSA might be produced by the activity of an iterative type I PKS such as ChlB1 involved in spirotetronate antibiotic chlorothricin biosynthesis pathway.[12] An alternative origin could involve the generation of salicylic acid from chorismate by a salicylate synthase[13] followed by an aromatic C-methylation leading to 6-MSA. The last step would be analogous, but at a different position of the aromatic ring, to C-methylation steps that take place during 3hydroxy-4-methyl-anthranilic acid biosynthesis, building block of the actinomycin chromophore,[14] or in 3,6-dimethylsalicylic moiety biosynthesis of enediyne maduropeptin.[15] Here we report the identification and characterization of the complete gene cluster for NAT biosynthesis from Streptomyces sp. Tü 6176. Gene inactivation and heterologous expression experiments provide evidence of possible functions of the identified genes. Furthermore, we show the production of additional natural benzoxazoles by Streptomyces sp. Tü 6176 including AJI9561 and novel 5hydroxynataxazole. Improving production yields of different benzoxazole compounds produced by Streptomyces sp. Tü 6176 is also reported.

Results Bioinformatic analysis of Streptomyces sp. Tü 6176 genome

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Genome sequence of nataxazole (NAT) producer was generated from 512,452 paired end sequences with a mean of 340.62 nucleotides, which gave a total of 174.55 Mb. De novo assembly of those sequences resulted in 1,265 contigs. N50 of the contig assembly was 10,531 bp, and the largest was around 68.1 Kb. Most of these contigs were ordered in 20 scaffolds. N50 of the scaffolding was 1,211,426 bp, and the largest scaffold was 3.5 Mb. The initial draft genome contains 8,820,073 nucleotides at 19.79-fold coverage, with a G+C content of 72.86 %. Genome annotation lead to identify 6,806 coding sequences, 1 rRNA operon and 67 tRNA loci. Analysis of Streptomyces sp. Tü 6176 genome sequence using bioinformatic tool antiSMASH 2.0 (antibiotics & Secondary Metabolite Analysis Shell)[16] predicted the existence of 38 secondary metabolite biosynthesis gene clusters (Table 1). Seventeen clusters contain modular enzyme coding genes such as PKSs (3 Type I, 1 Type II and 1 Type III), polyunsaturated fatty acid (PUFA) synthases (1 cluster), NRPSs (10 clusters) or hybrid PKS-NRPS (1 cluster). Despite the fact that most of precursors incorporated by PKS acyltransferase or NRPS adenylation domains can be predicted [17] it is hazardous to propose a presumptive structure for products derived from these clusters. In some cases the corresponding product has been predicted based in the similarity of deduced auxiliary proteins, other than PKS and NRPS, with known biosynthesis clusters (Table1). However, some of these clusters are highly conserved in other organisms and predictions could be more accurate. That is the case of cluster 37 (type III PKS) that might be involved in the production of 1,3,6,8-tetrahydroxynaphthalene (THN) and its auto-oxidation product flaviolin since this cluster is well conserved in several actinomycetes, particularly S. coelicolor A3(2), S. avermitilis MA-4680 and S. albus J1074.[18] Among NRPS clusters identified, two might determine the production of siderophore enterobactin (cluster 10) and zincophore coelibactin (cluster 25), based in

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their extreme similarity to the corresponding reported clusters from E. coli[19] and S. coelicolor A3(2).[20] Enterobactin has been previously shown to be produced by Streptomyces species.[21] Seven clusters are involved in the biosynthesis of other peptides and amino acidic compounds such as one hydroxamate siderophore, five ribosomal-derived peptides (3 bacteriocins and 2 lantipeptides) and ectoine. The remaining fourteen gene clusters might be involved in the biosynthesis of terpenes (8 clusters), butyrolactones (3 clusters), aminocyclitol hygromycin B, moenomycin-like phosphoglycolipid and melanin. Clusters corresponding to melanin (cluster 28), 5-hydroxyectoine (cluster 29) and

terpenes

albaflavenone

(cluster

1),

geosmin

(cluster

7),

hopene-

aminotrihydroxybacteriohopane (cluster 8), 2-methylisoborneol (cluster 18) and phytoene-isorerinatene (cluster 34) are also widely distributed among actinomycetes. Hygromycin B gene cluster would correspond to cluster 26 based in comparison to previously reported cluster (GenBank: AJ628642.1). It must be highlighted here that Streptomyces sp. Tü 6176 is resistant to hygromycin B up to 800 µg mL-1.

Identification of NAT biosynthesis gene cluster Taking in consideration all possible biosynthetic origins of NAT scaffolds, 3-HAA and 6-MSA, we concentrate on predicted clusters containing genes that could be involved in the biosynthesis pathways of 3-hydroxyanthranilic acid (3-HAA), 2,3-dihydroxybenzoic acid or salicylic acid from chorismate, and 6-methylsalicylic acid (6-MSA) by a type I PKS. Following those premises, a putative NAT biosynthesis cluster was initially identified by genome mining as cluster 3 (Table 1, Figure 1A). It contains anthranilate synthase (CF54_07345) and isochorismatase (CF54_07350) coding genes that should be involved in the biosynthesis of 3-HAA. In addition, a nonreducing iterative type I PKS,

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encoded by gene CF54_07385, was envisaged to be involved in the biosynthesis of 6MSA. Highly similar but hypothetical and uncharacterized clusters have been found in Streptomyces sp. PsTaAH-124 (Table 2) and Frankia EANpec1 (cluster FE20). The last one has been proposed to direct de biosynthesis of an unidentified aromatic compound.[22] To get a proof of the involvement of cluster 3 in NAT biosynthesis, gene natPK (CF54_07385) was inactivated using pnatPK leading to mutant SMPKS. Analysis of products accumulated by mutant SMPKS with respect to the wild type strain (Figure 1B), showed the disappearance of two peaks, one corresponding to NAT 1 and the second one 2 with characteristic benzoxazole absorption spectrum, UPLC retention time of 5.8 min and mass of m/z 417 [M+H]+. Structural elucidation of compound 2 showed it corresponds to novel benzoxazole 5-hydroxynataxazole (OH-NAT). This result clearly identified natPK as involved in the biosynthesis of NAT. Production of NAT and OH-NAT in mutant SMPKS was partially restored by introduction of pnatPKS containing natPK under the control of ermE*p promoter (Figure 1B). The 1.1 kb fragment inserted in pnatPK was used as a probe to screen a Streptomyces sp. Tü 6176 cosmid library. Nine cosmid clones were identified, four of them containing, in addition to natPK, anthranilate synthase (CF54_07345) and isochorismatase (CF54_07350) coding genes located in its vicinity at cluster 3. One of these cosmids, cos6E8 was envisaged to contain the whole NAT cluster. Cos6E8 insert consisting of 44.102 bp was de novo sequenced and annotated to complete the gaps present in NAT biosynthesis gene cluster identified from JFJQ01000000 (Figure 1A, Table 2).

In silico analysis and genetic characterization of NAT biosynthesis gene cluster

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6-MSA biosynthesis: The nonreducing iterative type I PKS NatPK is proposed to synthesize 6-MSA 4 for the biosynthesis of NAT 1 (Scheme 2A). It shows high level of identity and similarity with other iterative PKSs involved in 6-MSA biosynthesis such as those involved in polyketomycin,[23] PokM1, and chlorothricin,[12] ChlB1, biosynthesis. NatPK contains a characteristic iterative module composed of ketosynthase (KS), acyltransferase (AT), thioester hydrolase (TH), ketoreductase (KR) and acyl-carrier protein (ACP) domains, where TH might act as the 6-MSA-releasing domain.[24] NatPK would catalyze the assembly of one acetyl-CoA and three malonylCoAs to generate a tetraketide intermediate, which after a keto reduction at C-5 position, undergoes an intramolecular aldol condensation to generate 6-MSA. Expression of natPK in S. albus J1074, that lacks a 6-MSA synthase,[18b] using pnatPKS, leads to the accumulation of a compound with a retention time of 3.6 min, maxima of absorbance at 210, 242 and 307 nm, and a mass of 153 m/z [M+H]+ that would correspond to 6-MSA 4 (Figure 1C). Furthermore, this peak after purification and feeding to natPK mutant SMPKS, restored NAT production. In addition, NAT biosynthesis cluster contains a gene coding for a 4'phosphopantetheinyl (4’-PPT) transferase (natP, CF54_07330) that might be involved in post-translational modification of NatPK ACP, and other ACPs that might participate in the pathway (NatAC1 and NatAC2), for their correct functionality. However, there are at least four additional 4’-PPT transferase coding genes (CF54_16675, CF54_17390, CF54_17450 and CF54_07455) in Streptomyces sp. Tü 6176 genome, which might be also involved in this process. PPT transferase catalyzes the covalent linkage of coenzyme A 4’-PPT moiety to a conserved serine residue in the ACP domain. This 4’PPT linker functions as the carrier for the growing polyketide chain, in the case of PKSs.[25]

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3-HAA biosynthesis: The second scaffold required for NAT biosynthesis might be 3HAA (Scheme 2B). Four genes in the biosynthesis cluster encode enzymes that could be involved in the biosynthesis of this scaffold. A 3-deoxy-D-arabinose-heptulosonic 7phosphate (DHAP) synthase, encoded by natAL (CF54_07335), which shows similarity to EsmA6, CalB4 and TomC, involved in saphenamycin,[10] calcimycin[9] and tomaymycin[26]

biosynthesis,

respectively.

DHAP

synthases

condense

phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to yield DHAP, first intermediate in shikimate pathway leading to chorismate.[27] There is only one additional DHAP synthase gene (CF54_24340) in Streptomyces sp. Tü 6176 chromosome that might be involved in central metabolism shikimate pathway. Chorismate might be transformed into 2-amino-2-deoxyisochorismate (ADIC) by the activity of anthranilate synthase NatAN (CF54_07345). NatAN shares similarity with orthologue enzymes involved in saphenamycin, EsmA5,[10] calcimycin, CalB1,[9] and tomaymycin biosynthesis, TomD and TomP.[26] There are two additional anthranilate synthase coding genes (CF54_24325 and CF54_24750) in Streptomyces sp. Tü 6176, one of them probably involved in tryptophan biosynthesis. Next steps toward production of 3-HAA acid would be the conversion of ADIC into trans-2,3-dihydro-3-hydroxyanthranilate (DHHA), by isochorismatase NatIS (CF54_07350), and the transformation of DHHA to 3-HAA by 2,3-dihydro-2,3dihydroxybenzoate dehydrogenase NatDB (CF54_07355). Isochorismatase coding genes are also found in saphenamycin, EsmA4,[10]

and calcimycin, CalB2,[9]

biosynthesis pathways. Streptomyces sp. Tü 6176 contains several additional isochorismatase coding genes, one of them (CF54_07495) located in a NAT nearby NRPS cluster (cluster 5, Table 1) and another (CF54_12665) at putative enterobactin

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biosynthesis gene cluster (cluster 10, Table 1). A homologue to DHHA dehydrogenase NatDB is present in calcimycin biosynthesis pathway, CalB3.[9] In enterobactin cluster there is also a DHHA dehydrogenase encoded by CF54_12685.

Condensation of NAT biosynthetic scaffolds and tailoring: After production of NAT biosynthesis scaffolds, 6-MSA and 3-HAA, these should be activated and then condensed to form the final compounds in two steps (Scheme 2). Activation of 6-MSA might be carried out by natAC1 (CF5_07365), natL1 (CF5_07370) and natS (CF5_07375) encoded enzymes. NatAC1 shows similarity with 6-MSA ACP EsmD3 and contains core motif LGLSS present in ACPs (LGXDS) with an active serine residue.[28] NatL1 contains an AMP-binding domain (LSGGTTALPK) involved in ATP binding and amino acid adenylation that matches consensus core sequences described for this domain (LSGGxTxxxK)[29] and shows similarity to 6-MSA adenylase EsmD2. Streptomyces

sp.

Tü

6176

contains

several

additional

AMP-dependent

synthetase/ligases, one of them (CF54_12670) located in the enterobactin biosynthesis gene cluster (cluster 10, Table 1). NatS displays similarity with β-ketoacyl-ACP synthases including EsmD1, ChlB3, ChlB6 and PokM2. NatL1 might catalyze the adenylation of 6-MSA to generate AMP-6-MSA. Then, 6-MSA should be transfer to NatAC1 by NatS to generate a NatAC1-tethered 6-MSA group (Scheme 2A). The activation of 3-HAA scaffold might follow the same logic as described above and probably the same enzymatic activities. Alternatively a second enzyme that might catalyze the adenylation process is NatL2 (CF5_07380) that shows high similarity to coenzyme F390 synthetases from different origins: a phenylacetate-CoA ligase from Streptomyces sp. WK-5344 (BAM73635.1), bagremycin synthetase FevW, and a putative phenylacetate-CoA ligase, BagE, from Streptomyces sp. Tü 4128

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(AGM38213.1). Additionally, a second ACP is encoded by natAC2 (CF54_07410) located in downstream boundary of NAT biosynthesis cluster might serve as carrier of 3-HAA (Scheme 2B). Condensation of NatAC1-tethered 6-MSA and NatAC2-tethered 3-HAA might proceed, as it has been proposed for the generation of calcimycin benzoxazole ring,[9] by a nucleophilic attack from 3-HAA amino group to the terminal carbonyl group of NatAC1-tethered 6-MSA releasing the ACP NatAC1 (Scheme 2C). Then an acetalization reaction will close the heterocyclic ring generating the first NAT intermediate containing a benzoxazole moiety: NatAC2-tethered 6-methylcaboxamycin. Second benzoxazole moiety will follow the same reaction incorporating an additional 3HAA. However, we cannot discard the possible involvement of a free-standing 3-HAA in this process. In the first case, the resultant NAT intermediate, AJI9561, could be released from the ACP by the activity of natAM (CF54_07360) encoded enzyme. NatAM contains a conserved metal binding site characteristic of amidohydrolases, involving four histidines and one aspartic acid residue, as deduced by tridimensional structure of proteins of this family.[30] The amidohydrolase family includes metallodependent enzymes involved in hydrolysis of C-N and P-O bonds,[31] but also CS bonds.[32] To clarify the involvement and possible role of NatAM, NatAC1 and NatL1 in NAT biosynthesis a Streptomyces sp. Tü 6176 mutant, AM-L1, lacking natAM, natAC1 and natL1 was generated using pnatAM-L1. This mutant was unable to produce NAT and OH-NAT (Figure 2). Complementation of AM-L1 using pnatAML1-Se, which expresses the three genes deleted in the mutant strain, restored the production of both benzoxazoles, NAT and OH-NAT. On the other hand, partial complementation of AM-L1 using pnatAM-AC1t, which lacks natL1, did not restore

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benzoxazole production (Figure 2), confirming that natL1 encoding an AMP-dependent synthetase/ligase, is strictly required for the biosynthesis of both benzoxazoles, acting probably at early steps (Scheme 2A). Furthermore, partial complementation of AM-L1 using pnatAC1-St, which lacks natAM, did not restore benzoxazole production (Figure 2). This result indicates that natAM, encoding an amidohydrolase, is essential for NAT biosynthesis as is happens during pyrrolamides biosynthesis, where amidohydrolase Pya25 has been suggested to play a central role in controlling the flow of different intermediates into the following assembly line.[32b] Final step in NAT pathway is the carboxyl group O-methylation. However, no gene encoding an O-methyltransferase has been identified in NAT biosynthesis cluster. A hypothetical deduced protein might be encoded by natX, upstream of natPK, but no homology to known proteins has been observed. In order to verify its possible involvement in NAT pathway natX was expressed in Streptomyces sp. Tü 6176 under the control of ermE*p using pnatX. The recombinant strain generated was not able to produce higher amount of NAT than control strain carrying pEM4T. Furthermore, pnatX, pnatL2-X and pantL2-PK, were introduced into S. albus J1074. These strains, expressing natX, natL2-natX and natL2-natX-natPK respectively, were grown in R5A medium in an orbital shaker at 30ºC and 250 r.p.m. for t = 24, 48 or 72 h, then fed with AJ9561 (25 µg mL-1) and cultures extracted and analyzed 24 hours later. In the case of pantL2-PK, production of 6-MSA due to NatPK was observed, which verifies at least the last gene of the operon under the control of ermE*p was correctly expressed. However, in all of the bioconversion experiments transformation of AJ9561 to NAT was not observed, indicating that NatX is not the O-methyltransferase involved in NAT biosynthesis final step (Scheme 2C). On the other hand, an oxygenation is required to

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transform NAT into 5-hydroxynataxazole (Scheme 2C). No gene encoding such activity in NAT biosynthesis cluster or in its vicinity has been identified.

Regulation and secretion: Four putative regulatory genes are located in the vicinity of genes coding NAT structural enzymes described above. natR1 (CF54_07305) encodes a LuxR-family transcriptional regulator, containing a C-terminal LuxR type helix-turnhelix (HTH) DNA binding motif (PDOC00542), showing similarity to EsmT2 that controls the saphenamycin biosynthesis cluster,[10] Two TetR-family transcriptional regulators are encoded by natR2 (CF54_07325) and natR3 (CF54_07390), respectively. In both cases they share similarity with TetR regulators from different actinomycetes and contain the characteristic N-terminal HTH DNA binding domain signature characteristic of this family of regulators (pfam00440) that generally act as repressors.[33] The fourth regulatory gene is natR4 (CF54_07400/CF54_07405) that encodes a SARP-family transcriptional regulator that shares similarity to AfsR regulators from different actinomycetes and contain a characteristic OmpR-like DNAbinding domain[34] and a bacterial transcriptional activation domain (BTAD, pfam03704). In addition, natR4 contains a TTA that might imply an upper level of regulation triggering NAT biosynthesis pathway by some homolog to pleiotropic regulatory gene bldA encoding a tRNA-Leu.[35] The four regulatory genes located in NAT biosynthesis cluster were ectopically expressed into Streptomyces sp. Tü 6176 (Figure 3) using pnatR1, pnatR2, pnatR3 and pnatR4 leading to recombinant strains EER1, EER2, EER3 and EER4, respectively. Two regulatory genes, natR1 and natR4, were found to exert a positive effect over NAT production (Figure 3). In particular, expression of natR1 led to the accumulation of AJI9561 3 (UPLC retention time of 6.2 min and mass of 387 m/z [M+H]+) at t = 24 h

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(Figure 4B) when at the same time, control strain (Figure 4A) was not accumulating this compound. Early appearance of AJ9561 3 (t = 24 h) followed by a considerable reduction (t = 48 h) and subsequent accumulation of NAT 3 indicates that AJ9561 has been transformed into NAT. On the other hand, expression of regulatory genes natR2 or natR3 showed a negative effect on NAT 1 production that was reduced around 65 % in both cases (Figure 3). In all strains mentioned above, production of OH-NAT followed the same pattern of NAT (Figure 4) but showing lower variations. Four putative NAT transport protein coding genes are present in the biosynthesis cluster. Two ABC transport proteins are encoded by natT1 (CF54_ 07310) and natT2 (CF54_07315). NatT1 and NatT2 contain an N-terminal hydrophobic transmembrane domain with 5 and 6 transmembrane helices predicted, respectively. Furthermore, they contain a C-terminal ATP-binding domain composed by Walker A and B motifs characteristic of type III ABC transporters.[36] NatT1 and NatT2 might form a transport system with 2 transmembrane domains and 2 nucleotide binding domains forming a heterodimer.[36] On the other hand, natT3 encodes a MMPL domain (pfam03176)containing transport protein with 12 transmembrane helices predicted. NatR1, NatT1, NatT2 and NatT3 coding genes are located together and transcribed in the same direction, opposite to that of natR2 which could indicate the participation of NatT3 together with NatT1 and NatT2 in the efflux system. In addition, this gene arrangement might have implications for regulation of the transport process. Finally, natT4 (CF54_07395) encodes a protein containing 14 transmembrane helices predicted and similarity to transporters of major facilitator superfamily transporters (MSF, pfam07690) and EmrB/QacA-family of drug resistance transporters (TIGR00711). NatT4 shows similarity to lcz38 product from lactonamycin Z biosynthesis gene cluster in S. sanglieri.[37]

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Boundaries of nataxazole cluster: Upstream limit of NAT biosynthesis cluster might be located at orf-1 (CF54_07295) encoding a thioester reductase domain (IPR010080)containing protein that includes an NAD(P)-binding domain (IPR016040). This domain is present in α-aminoadipate reductase involved in lysine biosynthesis[38] but also forms part of NRPS enzymes such as MxcG, from Stigmatella aurantiaca, involved in siderophore myxochelin biosynthesis.[39] The rest of genes located upstream of orf-1 show no apparent relation with NAT biosynthesis: orf-2 (CF54_07290) contains an acetyl ornithine aminotransferase family domain (cd00610); orf-3 (CF54_07285) shows a polyketide cyclase/dehydrase and lipid transport domain (pfam03364); orf-4 (CF54_07280) encodes a SARP-family transcriptional regulator with similarity to SgvR3 and VmsS involved in the biosynthesis of antibiotics griseoviridin and viridogrisein in S. griseoviridis[40] and virginiamycin M in S. virginiae,[41] respectively; orf-5 encodes a putative sarcosine oxidase containing a NAD(P)-binding domain (IPR016040); and orf-6 encodes a putative short-chain dehydrogenase/reductase that contains a NAD(P)-binding domain (IPR016040). Since ornithine aminotransferases are involved in proline biosynthesis[42] and sarcosine oxidases catalyze the oxidative demethylation of sarcosine to yield glycine,[43] Orf-2 and Orf-5 might be involved in the biosynthesis of proline and glycine, respectively. Regarding the SARP regulator coding orf-4, it was ectopic expressed into Streptomyces sp. Tü 6176 under the control of constitutive promoter ermE*p using pSARP04, but no effect on NAT production was observed. Downstream limit of NAT biosynthesis gene cluster might be orf+1 (CF54_07420), first gene of cluster 4. Its deduced protein shows homology to PUFA synthase PfaA from different origins. Homologues to PfaABCD coding genes are all

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located in cluster 4 and have been shown in -proteobacteria to produce omega-3 polyunsaturated fatty acids.[44] To further clarify the limits between cluster 3 and 4, regulatory genes natR3 and natR4 (Figure 1A) were inactivated using pnatR4 and pnatR3, respectively, which led to mutant strains SMR3 and SMR4. Production of benzoxazoles by these strains was monitored for t = 7 days in R5A. SMR3 showed an early accumulation of AJI9561 and a final 4-fold improvement of NAT 1 production (Figure 5). On the other hand, in mutant SMR4 production of NAT was reduced approximately 57 % compared to Streptomyces sp. Tü 6176 (Figure 5). Both results are in concordance with those obtained by the ectopic expression of natR3 and natR4 into Streptomyces sp. Tü 6176 and confirm that both regulatory genes belong to NAT biosynthesis cluster (cluster 3).

Heterologous expression of nataxazole biosynthesis gene cluster In order to determine the boundaries of the NAT cluster and to identify the product of the pathway, heterologous production of a benzoxazole was attempted by expression of NAT biosynthesis cluster using pCAP01[45] derivative pNATAR (Figure 1A), which includes the proposed NAT biosynthesis gene cluster from natR1 to natAC2, generated by yeast transformation associated recombination (TAR) cloning. This construct was introduced into S. lividans JT46 leading to the production of AJ9561 3 at approximately 5 µg mL-1 (Figure 6), thus confirming that all genes required for the biosynthesis of AJI9561 are included in pNATAR but that the O-methyltransferase coding gene required for conversion of AJI9561 3 into NAT 1 and the oxygenase required to convert NAT 1 into OH-NAT 2 are not located in the biosynthesis gene cluster expressed. On the contrary, after several attempts, no S. albus J1074 clones carrying pNATAR were obtained. Interestingly, growth rate of S. lividans JT46/pNATAR was 40-50% lower

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than that of control strain S. lividans JT46/pCAP01. These results point to a possible effect of AJI9561 on S. albus and S. lividans growth. In order to test this possibility, two inactivated versions of NAT cluster were generated from pNATAR carrying a deletion in natAM (pNATAM∆) or natPK (pNATPKS∆), both therefore unable to produce any benzoxazole. As expected, expression of pNATAM∆ in S. lividans JT46 led to the accumulation of 6-MSA 4, while S. lividans JT46 carrying pNATPKS∆ was unable to produce 6-MSA and AJ9561 (Figure 6). In both cases, these strains were growing at same rates as S. lividans JT46/pCAP01. Same results were observed in S. albus J1074 carrying pNATAM∆ or pNATPKS∆.

Biological activity of AJI98561, NAT and OH-NAT Antibiotic activity of AJI9561, NAT and OH-NAT was tested via antibiotic disc diffusion assay against S. lividans JT46 and S. albus J1074, both used for heterologous expression of NAT cluster. While NAT and OH-NAT showed no antibiotic activity, AJI9561 was clearly active against S. lividans JT46 above 5 µg and against S. albus J1074 above 1 µg. Antibiotic activity of NAT and OH-NAT and AJI9561 were tested also against Micrococcus luteus, Escherichia coli, and yeast Candida albicans. Neither NAT nor OH-NAT showed antibacterial or antifungal activity. On the other hand, AJI9561 was active against M. luteus above 5 µg but showed no activity against E. coli and C. albicans. Cytotoxicity of NAT and OH-NAT against human tumor cell lines HT29, A549, MDA-MB-231, AGS and A2780, including mouse cell line NIH/3T3 used as control to evaluate cytotoxicity against non malignant cells, was moderate in the range of 10 µM (IC50). No statistical differences in activity were observed between NAT and OH-NAT. Cytotoxic activity of AJI9561 has been previously reported.[3]

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Discussion Streptomyces sp Tü 6176 NAT biosynthesis gene cluster has been identified in silico by genome mining. This cluster consists of 21 genes: 12 coding for structural proteins, 4 for regulatory proteins, 4 probably involved in NAT secretion and 1 with unknown function. These genes are organized in at least six transcriptional units and encode all activities required for AJI9561 biosynthesis. The remaining activity, an Omethyltransferase, which would convert AJI9561 into NAT, is not apparently encoded by any gene of the cluster. In addition, gene coding the oxygenase required to transform NAT into novel benzoxazole OH-NAT is also absent from the cluster. Location of structural genes outside of a gene cluster is not a common feature for secondary metabolite pathways in actinomycetes but some examples have been previously reported.[46] Confirmation of the involvement of this cluster in NAT biosynthesis was achieved by inactivation of type I PKS coding natPK that led to mutant SMPKS unable to produce NAT and OH-NAT since biosynthesis of 6-MSA had been abrogated. On the other hand, heterologous expression of NAT cluster into S. lividans JT46 led to the production of AJ9561, which confirms that the O-methyltransferase activity converting AJI9561 into NAT and that converting NAT into OH-NAT are located outside of the cluster. Furthermore, the high antibiotic activity exerted by AJI9561 against S. lividans and, in particular, S. albus, strongly suggests this O-methyltransferase activity might act as a possible resistance mechanism in Streptomyces sp. Tü 6176. The biosynthesis pathway of NAT links primary metabolism shikimate pathway to secondary metabolism (polyketides) to generate a benzoxazole bioactive compound. Shikimate pathway, required for the biosynthesis of NAT precursor 3-HAA, forms part of aromatic amino acids biosynthesis, which is highly regulated at different levels. One

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of the regulation checkpoints is the activity of DAHP synthase, first step of shikimate pathway, which leads to chorismate. The activity of this enzyme is regulated by feedback inhibition of the final product. In E. coli there are three different DAHP synthases each one sensitive to one aromatic amino acid: L-phenylalanine (Phe), Ltyrosine (Tyr) and L-tryptophan (Trp). In actinomycete Corynebacterium glutamicum there are only two DAHP synthases, sensitive to Tyr and Phe, respectively.[27] In the case of S. coelicolor A3(2), it contains two DAHP synthase coding genes: sco2115 encodes a tryptophan sensitive enzyme that might be involved in aromatic amino acids biosynthesis,[47] while the second (sco3210) is located in calcium-dependent antibiotic (CDA) biosynthesis gene cluster.[20] The main reason of this DAHP synthase might be the supply of chorismate for tryptophan biosynthesis (two Trp moieties are present in the CDA structure) without feedback inhibition by the final product. The same biosynthetic model might be followed in Streptomyces sp. Tü 6176 for NAT biosynthesis and other chorismate-dependent secondary metabolites. In this organism, DHAP synthase NatAL might be involved in secondary metabolism, and probably is not regulated by aromatic amino acids. The enzyme encoded by cf54_24340 should be involved in primary metabolism and for instance feedback regulated by, at least one, aromatic amino acid. A second regulatory checkpoint for the biosynthesis of Trp is the feedback inhibition of anthranilate synthase.[48] Probably, to avoid this regulatory system the NAT biosynthesis cluster contains a pathway specific anthranilate synthase, NatAN, to provide 3-HAA for secondary metabolism benzoxazole biosynthesis. In addition to a metabolic control bypassed by the presence of specific activities dedicated to NAT biosynthesis, NatAL and NatAN, NAT production is directly regulated by four pathway-specific regulatory genes located in the cluster. Two of them, natR2 and natR3, encode transcriptional repressors of the TetR family that under

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ectopic expression led to reduced levels of NAT. On the other hand, inactivation of natR3 led to improve NAT production starting by an early accumulation of AJ9561. There are two additional pathway-specific regulatory genes, natR1 and natR4, which act as transcriptional activators. Ectopic expression of each of them led to improvements of NAT production up to 4-fold. In particular, expression of natR4 induced an early increment of NAT intermediate AJI9561. Considering the increased production of AJI9561 in strains EER4 and SMR3 it might be plausible that NatR3 and NatR4 are involved in the onset of NAT biosynthesis. In conclusion, Streptomyces sp. Tü 6176, originally described as producer of NAT, is capable of producing at least three related benzoxazole compounds: AJI9661, NAT and OH-NAT by an apparently simple biosynthesis pathway but that otherwise is highly regulated. In addition, production of NAT and OH-NAT might be metabolically linked to other pathways from primary or secondary metabolism since the genes encoding those activities required for their biosynthesis are absent from AJI9561 biosynthesis gene cluster. This work sets the stage to explore those metabolic links and the generation of novel benzoxazole compounds.

Experimental Section Strains, culture conditions and plasmids Bacterial strains used in this work were: Streptomyces sp. Tü 6176 producer of NAT;[1] S. albus J1074,[49] and S. lividans JT46[50] were used for heterologous expression; E. coli DH10B (Invitrogen), LE392MP (Epicentre) and ET12567 (pUB307)[51] were used for subcloning and intergeneric conjugation, respectively; E. coli LE392MP (Epicentre)

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was used to propagate a cosmid library; and yeast strain Saccharomyces cerevisiae VL6-48 (MAT alpha, his3-D200, trp1-D1, ura3-52, lys2, ade2-101, met14, psi+cir0) was used for TAR cloning. Growth medium for Streptomyces sp. Tü 6176 and S. albus was tryptone soya broth (TSB), MA medium was used for sporulation and R5A as production medium.[52] E. coli media were those described in the literature.[53] For production and purification of benzoxazoles Streptomyces Tü 6176 was grown in GHSA medium: MOPS (21 g L-1), glucose (10 g L-1), yeast extract (0.5 g L-1), MgSO4·7H2O (0.6 g L-1), soy flour (10 g L-1), R5A oligoelementens (2mL L-1), pH 6.75. Yeast transformants were selected on YNB/sorbitol-trp medium: yeast nitrogen base without amino acids (0.17 %), yeast synthetic drop-out medium supplements without tryptophan (0.19 %), sorbitol (1 M), D-glucose (2 %), adenine (100 mg L-1) and agar (2 %); and YNB-ura media: yeast nitrogen base without amino acids (0.17 %), yeast synthetic drop-out medium supplements without uracile (0.19 %), D-glucose (2 %), adenine (100 mg L-1) and agar (2 %). Yeast colonies were cultured overnight in YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose, 100 mg L-1 adenine). When plasmid-containing clones were grown, the medium was supplemented with appropriate antibiotics: ampicillin (100 µg mL-1) tobramycin (20 µg mL-1), apramycin (25 µg mL-1) thiostrepton (50 µg mL-1) tetracycline (10 µg mL-1), chloramphenicol (25 µg mL-1) or nalidixic acid (50 µg mL-1). Plasmids used in this work were pOJ260[54] for gene disruption and pEFBAoriT[55] and pHZ1358[56] for gene replacement. pEM4T[57] and pSET152[54] were used for gene expression. Yeast/E. coli shuttle-actinobacterial chromosome integrative vector pCAP01 was used for TAR cloning.[45] pCXJ18[58] was the source of URA3 gene marker. pSL1180 (Amersham) was used for regular cloning and pCR-BLUNT (Invitrogen) for cloning PCR products.

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DNA manipulation DNA manipulations were performed according to standard procedures for E. coli[53] and Streptomyces.[51] PCR amplifications were conducted by using Herculase II Fusion DNA Polymerase (Agilent Technologies) with a touchdown PCR procedure. When needed dimethylsulphoxide (DMSO) 2.5 % was used. Termocycler (SureCycler 8800, Agilent Technologies) was programmed as follow, initial denaturation at 99.9 ºC for t = 4 min; 20 cycles of 99.9 ºC for t = 20 sec, 65-45 ºC touchdown for t = 20 sec and 72 ºC for t = 45 sec followed by 10 cycles of 99.9 ºC for t = 20 sec, 60 ºC for t = 20 sec and 72 ºC for t = 45 sec. Final extension was performed at 72 ºC for t = 3 min. PCR products of the expected size were initially cloned into pCR-BLUNT for sequencing verification. All oligoprimers used for PCR amplifications are shown in Table S1 (Supporting information). A cosmid library of Streptomyces sp. Tü 6176 genomic DNA was constructed. DNA fragments obtained from a partial digestion with Sau3AI were ligated to cosmid pWE15 digested with BamHI and in vitro packaged using Gigapack III Gold packaging Extract kit according to the manufacturer’s handbook (Stratagene). The resulting E. coli transductants were picked and transferred to 96-well microtitre plates containing Luria broth (LB) medium and ampicillin. Clones were replica plated onto Luria agar (LA) plates containing ampicillin. After overnight growth at 37oC, colonies were transferred to nylon membrane filters for in situ colony hybridization analysis according to published methods[53] and screened using labeled probes that were generated using DIG DNA labeling and detection kit (Roche).

Construction of vectors

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For gene expression purposes two pSET152 derivatives, containing constitutive ermE*p,[59] were generated. Plasmid pSETec was generated by subcloning a NheI-XbaI fragment, containing the ermE*p, from pEM4T into pSET152 digested XbaI. On the other hand, thiostrepton resistance gene tsr was amplified by PCR from pEM4T using oligoprimers Tsrfw and Tsrrv. The resultant fragment was digested XbaI and subcloned into pSETec digested NheI leading to pSETeTc.

Construction of plasmids for gene inactivation Gene disruption of natPK was accomplished using pnatPK. For this purpose a natPK internal fragment of 1100 bp was amplified by PCR using primers PKS1Ta and PKS1Tb and Streptomyces sp. Tü 6176 chromosomal DNA as template. The resultant fragment was digested EcoRI-HindIII and cloned into pOJ260 digested with the same enzymes. Construct pnatPK was introduced into Streptomyces sp. Tü 6176 by intergeneric conjugation form E. coli ET12567 (pUB307) leading to mutant strain SMPKS. Deletion of natAM, natAC1 and natL1 region and its replacement by acc(3)IV was accomplished using pnatAM-L1. Two DNA fragments flanking natAM-L1 region were amplified by PCR using cos6E8 as template. Fragment A of 2.1 kb, amplified using oligoprimers READHL-1 and READHL-2, was cloned into BamHI-EcoRV digested pEFBAoriT leading to pREAD-A. Fragment B of 1.6 kb, amplified using oligoprimers READHL-3 and READHL-4, was cloned into SpeI-NsiI digested pREASA, leading to pREAD-AB. Afterwards, a 6 kb SpeI-EcoRV fragment from pREAS-AB was cloned into pHZ1358. For deletion of natR3 pnatR3 was generated by amplifying two 2 kb fragments using primer pairs RETetR27-1/RETetR27-2 and RETetR27-3/RETetR27-4. Both

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fragments were subcloned sequentially into pEFBAoriT and then transferred to pHZ1358 as described above. Gene replacement of natR4 was accomplished following the same method by amplifying two 2.1 kb fragments using primer pairs RESARP291/RESARP29-2 and RESARP29-3/RESARP29-4. Both fragments were subcloned sequentially into pEFBAoriT and then transferred to pHZ1358 as described above, leading to pnatR4. Constructs pnatAM-L1, pnatR3 and pnatR4 were introduced into Streptomyces sp. Tü 6176 by intergeneric conjugation form E. coli ET12567 (pUB307), leading to mutant strains AM-L1, SMR3 and SMR4, respectively. In these cases an apramycin and thiostrepton resistant mutant, obtained by a single-crossover event, was grown in the absence of antibiotics and then screened for the lost of thiostrepton

resistance,

keeping apramycin

resistance because

of a double

recombination event.

Construction of plasmids for gene expression All genes tested were expressed under the control or ermE*p. To express natPK pnatPKS was used. Oligoprimers PKSCOMP-1 and PKSCOMP-2 were used to amplify a 6 kb fragment, containing natPK, which was digested BamHI-EcoRI and cloned into BamHI-EcoRI digested pEM4T. For the expression of natX, it was amplified as a 600 bp fragment using oligoprimers 1525fw and 1525rv, then digested BamHI-EcoRI and cloned into pEM4T, leading to pnatX. Oligoprimers COA1126A and COA318F were used to amplify a 2.5 kb fragment, containing natL2 and natX, which was digested BamHI-EcoRI and cloned into pEM4T leading to pnatL2-X. Primers 1524-PKSfw and PKSCOMP-2 were used to amply a 8 kb fragment, containing natL2, natX and natPK, which was cloned into BamHI-EcoRI digested pEM4T leading to pnatL2-PK. For the expression of natAC1 and natAM pnatAM-AC1t was constructed by amplification of a

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1.8 kb fragment using primers 1519fw and ACPrv that was digested EcoRV-EcoRI and cloned into EcoRV-EcoRI pSETeTc. For the expression of natS, natL1 and natAC1 a 3 kb PCR fragment, obtained using oligoprimers PAMACup and PAMACrp, was digested BamHI-EcoRI and cloned into pSETeTc leading to pnatAC1-St. For the expression of natAM, natAC1, natL1 and natS, pnatAM-L1-Se was constructed by amplifying a 4.4 kb fragment using primers 1519fw and PAMACrp that was digested MfeI-EcoRI and cloned, in the right orientation, into EcoRI digested pSETec, leading to pnatAM-L1-S. The final construct was generated by cloning ermE as a NheI-AvrII fragment obtained for pAGE into pnatAM-L1-S. Regulatory genes located in cos6E8, orf-4, natR1, natR2, natR3 and natR4, these were amplified using primer pairs SAR4fw/SAR4rv, LuxR9fw/LuxR9rv, TetR14fw/TetR14rv, TetR27fw/TetR27rv and SAR29fw/SAR29rv, which lead to 1248 bp, 3057 bp, 948 bp, 934 bp and 2658 bp fragments, respectively. Fragments containing orf-4 or natR3 were digested BamHIEcoRI and cloned into pSETec digested with the same enzymes to obtain pSARP04 and pnatR3, respectively. Fragments containing natR2 or natR4 were digested BglII-EcoRI and cloned into pSETec digested with the same enzymes to obtain pnatR2 and pnatR4, respectively. Finally, natR1 was cloned as a EcoRV-EcoRI into pSETec leading to pnatR1. pEM4T, pnatPKS, pnatX, pnatL2-PK, pSETeTc, pnatAM-AC1t, pnatAC1-St, pnatAM-L1-S, pnatAM-L1-Se, pSETec, pSARP04, pnatR1, pnatR2, pnatR3 and pnatR4 were introduced in the appropriate Streptomyces strains by intergeneric conjugation form E. coli ET12567 (pUB307) and transconjugants were selected for resistance to thiostrepton or apramycin depending on each plasmid.

Expression of the nataxazole biosynthesis gene cluster

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NAT biosynthesis cluster (natR1 to natAC2), for heterologous expression into S. albus J1074 and S. lividans JT46, was captured by TAR cloning, essentially performed as previously described.[45] Arms required to capture the cluster were generated by PCR amplification of two pathway flanking regions of approximately 1kb with primer pairs SpeI-Nup/BamHI-Nup and BamHI-Nlow/XhoI-Nlow, which contain the indicated restriction sites. Capture arms were then digested with BamHI and ligated. The resulting assembled fragment was amplified with primers SpeI-Nup/XhoI-Nlow and cloned in the same sites of pCAP01 to produce capture plasmid pCAPNAT. Yeast strain VL6-48 was then transformed as previously described[60] with pCAPNAT (0.5 µg) linearized by digestion with BamHI and with construction cos6E8 (0.5 µg). Positive yeast colonies were cultured overnight for glass-beads extraction[61] of the resulting vector (pNATAR) containing the nataxazol pathway. Plasmids from three independent yeast colonies were then subjected to physical characterization of the nataxazol gene cluster by digestion with NcoI. The verified pNATAR was transferred to Streptomyces by protoplast transformation.[51] Deletion of natAM or natPK genes from pNATAR was accomplished by yeast in vivo homologous recombination-mediated PCR targeting.[62] URA3 gene marker was amplified from pCXJ18 by PCR with primer pairs mutAMd/mutAMrv and mutPKd/mutPKrv, which contained 20 base pairs complementary to URA3 plus flanking regions of about 40 bases complementary to natAM and natPK genes respectively. PCR fragments were purified and transformed (0.5 µg) by LiAc/SS carrier DNA/PEG method[63] into S. cerevisiae VL6-48 carrying pNATAR to replace target genes by URA3. The resulting constructs pNATAM∆ and pNATPKS∆ lacking natAM and natPK respectively were then isolated from three independent yeast colonies selected on YNB-ura medium. Constructs were introduced into E. coli and purified to

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confirm the gene replacements by PCR. The verified pNATAM∆ and pNATPKS∆ were transferred into Streptomyces by protoplast transformation.[51]

DNA sequencing and analysis Streptomyces sp. Tü 6176 chromosome: Genome sequence of NAT producer was generated at Lifesequencing (Valencia, Spain) by pyro-sequencing technology using the 454 Life Science-Roche platform followed by a de novo assembly using parameters by default in Newbler assembler software version 2.8.[64] Annotation was performed using PGAAP pipeline.[65] Computer-aided database searching and sequence analysis were carried out using bioinformatic tool antiSMASH[16] and BLAST program.[66]

Cosmid cos6E8: NAT biosynthesis gene cluster was located in cosmid cos6E8. DNA sequencing of this region was generated at Macrogen Europe (Amsterdam, the Netherlans). Annotation of this sequence was generated manually by computer-aided database searching and sequence analysis performed with BLAST program.[66] Analysis of PKS predicted proteins were carried out using program ASMPKS[67] and analysis of transmembrane regions in putative membrane proteins were performed using program TMHMM v. 2.0.[68]

Nucleotide sequence accession numbers: Streptomyces sp. Tü 6176 Whole Genome Shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession number JFJQ00000000. The version described in this paper is the first version, JFJQ01000000 and consists of sequences JFJQ01000001 to JFJQ01001265. Sequence of

cosmid

cos6E8

containing

NAT

cluster

has

DDBJ/EMBL/GenBank under the accession number LN713864.

been

deposited

in

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Analysis of metabolites by UPLC and LC-MS Whole cultures of selected strains or mutants were extracted with ethyl acetate containing formic acid (1 %) (to enhance the extraction of compounds containing ionizing groups) and analyzed by reversed phase chromatography in an Acquity UPLC instrument fitted with a BEH C18 column (1.7 µm, 2.1 x 100 mm, Waters), with acetonitrile and trifluoroacetic acid (TFA) (0.1 %) as solvents. Samples were eluted with acetonitrile (10 %) for t = 1 min, followed by a linear gradient of acetonitrile (10 % to 100 %) over t = 7 min, at a flow rate of 0.5 mL min-1 and a column temperature of 35ºC. For HPLC-MS analysis, an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 µm, 2.1 x 150 mm, Waters) was used. Solvents were the same as above and elution was performed with an initial isocratic hold with acetonitrile (10 %) during t = 4 min followed by a linear gradient of acetonitrile (10 % to 88 %) over t = 30 min, at 0.25 mL min-1. MS analysis were done by electrospray ionization in the positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 V. Detection and spectral characterization of peaks was performed in both cases by photodiode array detection at 330 nm, using Empower software (Waters) to extract bidimensional chromatograms at different wavelengths, depending on the spectral characteristics of the desired compound. Production cultures were incubated at 30 ºC and 250 r.p.m. for t = 7 days and then 1 mL samples from each of the wells were extracted with an equal volume of ethyl acetate in order to quantify compound levels in the cultures.

Isolation of compounds from Streptomyces Tü 6176

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Nataxazole and 5-hydroxynataxazole were isolated from Streptomyces sp. Tü 6176 grown at 30ºC during t = 7 days in 80 plates of GHSA solid medium (2 L). Plates were inoculated with a Streptomyces sp. Tü 6176 culture grown in TSB (100 µL), in an orbital shaker at 30ºC and 250 r.p.m. during t = 24 h. AJ9561 was isolated from mutant strain SMR3 grown in 40 Erlenmeyer flasks (250 mL), each containing R5A (50 mL), in an orbital shaker at 30 ºC and 250 r.p.m. during 24 hours. Each flask was inoculated with a seed culture of SMR3 culture grown in TSB in an orbital shaker at 30 ºC and 250 r.p.m. during t = 24 h. Compounds were extracted from solid medium twice with ethyl acetate acidified with formic acid (1 %). From liquid medium, cultures were centrifuged, the supernatants were discarded and the pellets were extracted twice with ethyl acetate acidified with formic acid (1 %). Organic extracts were evaporated in vacuo and the resulting dry extracts were redissolved in 5 ml of a mixture of DMSO and methanol (1:1). Compounds of interest were purified by preparative HPLC using a SunFire C18 column (10 µm, 10 x 250 mm, Waters). Compounds were chromatographed with mixtures of acetonitrile and TFA (0.05 %) in water in isocratic conditions optimized for each peak, at 7 mL min-1.

Structural characterization of compounds Compound 2 corresponding to 5-hydroxynataxazole (OH-NAT) was subjected to LC/ESI-TOF analysis in order to determine its molecular formula. Structural elucidation of OH-NAT was carried out by analysis of a combination of 1D (1H and

13

C), and 2D

(1H-1H COSY, TOCSY, 1H-13C heteronuclear single-quantum correlation (HSQC)edited and 1H-13C heteronuclear multiple-bond correlation (HMBC)) NMR experiments and comparison of the spectra obtained with those described in the literature

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(supplementary data). Solvent used in the NMR analyses was deuterated DMSO (DMSO-d6) (Figure S1 and Table S2, Supporting information).

Bioactivity testing Antibiotic activity of 5-hydroxynataxazole and AJI9561 were analyzed via antibiotic disc diffusion assay against S. albus J1074, S. lividans JT46, M. luteus and E. coli. Antifungal activity of 5-hydroxynataxazole was tested against C. albicans. AJI9561 was analyzed via antibiotic disc diffusion assay against S. albus J1074 and S. lividans JT46. In all cases 1 µg, 5 µg, 10 µg and 20 µg of each compound were used. Plates were incubated for t = 5 days at 30 °C. Antitumor activity of 5-hydroxynataxazole was tested against the following human tumor cell lines: colon adenocarcinoma (HT29), non-small cell lung cancer (A549), breast adenocarcinoma (MDA-MB-231), gastric carcinoma (AGS), and ovarian carcinoma (A2780). Mouse embryonic fibroblast cell line NIH/3T3 was used as control to evaluate cytotoxicity against non malignant cells. Quantitative measurement of cell growth and viability at t = 24, 48 and 72 h was carried out by using a colorimetric type of assay, using the cell proliferation and cytotoxicity assay system Cell Counting Kit-8 (CCK-8) following manufacturer protocol (Dojindo Molecular Technologies, Inc). Acknowledgments We like to thank Dr. Fernando Reyes from Fundación Medina for technical support in structural elucidation of compounds. We thank Bradley S. Moore (UCLA, San Diego) for gift of pCAP01. We also thank Natalay Kouprina (NCI, USA) for valuable comments on TAR cloning procedure and for kindly provide the yeast strain. This research was supported by a project granted by the Spanish Ministry of Economy and Competitiveness (MINECO) (BIO2012-33596 to J.A.S.). C.C.P. was the recipient of a

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predoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (MINECO). A.A.L. was the recipient of a predoctoral fellowship of FICYT (Asturias). We thank Obra Social Cajastur for financial support to C.O.

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Legends for figures and schemes Scheme 1. Chemical structure of nataxazole and related benzoxazole compounds.

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Scheme 2. Proposed pathway for nataxazole biosynthesis. A) Biosynthesis of precursor 6-methylsalicilic acid (6-MSA 4) and its activation. B) Biosynthesis of precursor 3hydroxyanthranilic acid (3-HAA) and its activation. C) Condensation of 6-MSA and 3HAA for the production of AJI9561 3 and its conversion into nataxazole 1 and 5hydroxynataxazole 2. ADIC, 2-amino-2-deoxyisochorismate; DHAP, 3-deoxy-Darabinose-heptulosonic 7-phosphate; DHHA, 2,3-dihydro-3-hydroxyanthranilate; E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate.

Figure 1. A) Genetic organization of the nataxazole gene cluster in Streptomyces sp. Tü 6176 from cosmid cos6E8. Region present into pNATAR is represented by a bar. B) UPLC analysis of Streptomyces sp. Tü 6176 wild-type (WT), mutant SMPKS, SMPKS carrying pEM4T (SMPKS/pEM4T) and SMPKS expressing natPK (SMPKS/pnatPKS) extracts obtained from cultures grown in complex medium R5A during t = 7 days. C) UPLC analysis of organic extracts from S. albus J1074 carrying pEM4T (control) and pnatPKS (expressing natPK). Compounds: nataxazole 1, 5-hydroxynataxazole 2 and 6methylsalicylic acid 4.

Figure 2. UPLC analysis of Streptomyces sp. Tü 6176 wild-type (WT), mutant AML1, AM-L1 carrying pnatAM-L1Se, AM-L1carrying pnatAM-AC1t and AM-L1 carrying pnatAC1-St extracts obtained from cultures grown in complex medium R5A during t = 7 days. Compounds: nataxazole 1 and 5-hydroxynataxazole 2.

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Figure 3. Time-course of nataxazole production by Streptomyces sp. Tü 6176 strains expressing pSETec (Control), pnatR1 (EER1), pnatR2 (EER2), pnatR3 (EER3) and pnatR4 (EER4). Production experiments were performed by triplicate in R5A.

Figure 4. A) UPLC analysis of benzoxazoles AJI9561 3, nataxazole 1 and 5hydroxynataxazole 2 produced by Streptomyces sp. Tü 6176 harboring pSETec (Control) grown in R5A. B) UPLC analysis of benzoxazoles AJI9561 3, nataxazole 1 and 5-hydroxynataxazole 2 produced by Streptomyces sp. Tü 6176 harboring pnatR1 (EER1) grown in R5A.

Figure 5. UPLC analysis of Streptomyces sp. Tü 6176 wild-type (WT) and mutants SMR3 and SMR4 extracts obtained from cultures grown in R5A during t = 7 days.

Figure 6. UPLC analysis of extracts obtained from cultures of S. lividans JT46 carrying pCAP01, pNATAR, pNATAM∆ or pNATPKS∆, grown in R5A during t = 7 days, showing the production of AJI9561 3 or 6-methylsalicylic acid 4.

Tables Table 1. Secondary metabolite biosynthesis gene clusters identified in Streptomyces sp. Tü 6176 by genome mining.

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Cluster 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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Location CF54_[a] 03620-03625 07050-07095 07305-07410 07415-07460 07485-07515 08595-08645 08770 10925-10955 11885-11915 12645-12705 13990-14130 14140-14185 14730-14780 14900-15020 15025 15055-15110 15465-15490 15655-15660 16410-16710 17350-17370 17425-17550

Type Terpene Siderophore Type I PKS PUFA NRPS Bacteriocin Terpene Terpene Bacteriocin NRPS Phosphoglycolipid Lantipeptide Bacteriocin NRPS Terpene NRPS Lantipeptide Terpene NRPS Butyrolactone NRPS-PKS

Predicted product Albaflavenone Unknown Nataxazole Unknown Unknown Unknown Geosmin Hopene Unknown Enterobactin Moenomycin-like Unknown Unknown Azinomycin B-like Unknown coelichelin-like Unknown 2-methylisoborneol Unknown Unknown Virginiamycin M-like Pristinamycin-like 22 17570-17915 Type I PKS Vicenistatin-like 23 18585-18595 Butyrolactone Unknwon 24 19785-19880 Type I PKS Unknwon 25 20670-20720 NRPS Coelibactin 26 20835-20965 Aminocyclitol Hygromycin B 27 21030-21050 NRPS Unknown 28 21230-21235 Melanin Melanin 29 25565-25595 Ectoine synthase 5-Hydroxyectoine 30 26680-26810 NRPS Unknown 31 29075-29100 NRPS Unknown 32 29305-29390 NRPS Unknown 33 29415-29640 Terpene Longestin-like 34 29990-30015 Terpene Phytoene-Isorerinatene 35 30170-30215 Butyrolactone Unknown 36 33430-33490 Type II PKS Putative spore pigment 37 34275-34280 Type III PKS THN, flaviolin 38 34650-34655 Terpene Unknown [a] Location and predicted products based on antiSMASH analysis of Streptomyces sp. Tü 6176 genome JFJQ01000000.

Table 2. Deduced proteins from cos6E8 sequence containing the nataxazole biosynthesis cluster. Gene

Location

aa[a]

Proposed Function

Closest similar protein (%

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orf-6

CF54_ NA[b]

176

orf-5

NA[b]

352

Short-chain dehydrogenase/reductase Sarcosine oxidase

orf-4

07280

333

SARP-family regulator

orf-3

07285

139

Putative cyclase/dehydrase

orf-2

07290

413

Ornithine aminotransferase

orf-1

07295

385

natR1

07305

935

Thioester reductase domaincontaining protein LuxR-family regulator

natT1

07310

579

ABC transporter

natT2

07315

608

ABC transporter

natT3

NA[b]

722

natR2

07325

198

natP

07330

236

natAL

07335

460

natAN

07345

636

natIS

07350

230

natDB

07355

274

natAM

07360

494

natAC1

07365

89

natL1

07370

539

natS

07375

350

natL2

07380

436

natX natPK

NA[b] 07385

195 1717

natR3

07390

232

MMPL domain-containing transport protein Nataxazole biosynthesis, TetR-family regulator Nataxazole biosynthesis, 4’-PPT[d] transferase Nataxazole biosynthesis, DHAP[e] synthase, Nataxazole biosynthesis, Anthranilate synthase Nataxazole biosynthesis, Isochorismatase Nataxazole biosynthesis, DHHA[f] dehydrogenase Nataxazole biosynthesis, Amidohydrolase Nataxazole biosynthesis, ACP Nataxazole biosynthesis, AMP-dependent synthetase/ligase Nataxazole biosynthesis, 3-oxoacyl-ACP synthase III Nataxazole biosynthesis, coenzyme F390 synthetase Deduced protein Nataxazole biosynthesis, 6-methylsalicylic acid synthase Nataxazole biosynthesis, TetR-family regulator

[c]

Identity / Similarity) origin WP_018564658.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564657.1 (99/100) Streptomyces sp. PsTaAH-124 WP_018564656.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564655.1 (98/98) Streptomyces sp. PsTaAH-124 WP_018564654.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564653.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564652.1 (99/100) Streptomyces sp. PsTaAH-124 WP_018564651.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564650.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564649.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564648.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564647.1 (98/98) Streptomyces sp. PsTaAH-124 WP_018564646.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564645.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564644.1 (99/100) Streptomyces sp. PsTaAH-124 WP_018564643.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564642.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564641.1 (99/100) Streptomyces sp. PsTaAH-124 WP_018564640.1 (99/100) Streptomyces sp. PsTaAH-124 WP_018564639.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564638.1 (100/100) Streptomyces sp. PsTaAH-124 No significant homology found WP_018564637.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564636.1 (100/100) Streptomyces sp. PsTaAH-124

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natT4

07395

515

natR4

845

natAC2

07400 07405 07410

orf+1

07415

957[c]

88

Nataxazole biosynthesis, EmrB/QacA-family transporter Nataxazole biosynthesis, SARP-family regulator ACP

Polyunsaturated fatty acid synthase [a] aa, aminoacids of the deduced protein. [b] NA, not annotated in JFJQ01000000. [c] Incomplete. [d] 4’-PPT, 4'-phosphopantetheinyl. [e] DHAP, 3-deoxy-D-arabinose-heptulosonic 7-phosphate. [f] DHHA, 2,3-dihydro-3-hydroxyanthranilate

WP_018564635.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564634.1 (99/99) Streptomyces sp. PsTaAH-124 WP_018564633.1 (100/100) Streptomyces sp. PsTaAH-124 WP_018564632.1 (99/99) Streptomyces sp. PsTaAH-124

Text suggestion for the table of contents Streptomyces sp. Tü 6176 produces cytotoxic benzoxazole nataxazole and its derivative 5-hydroxynataxazole.

In

certain

conditions

benzoxazole

AJI9561,

nataxazole

intermediate, is accumulated. Nataxazole biosynthesis requires shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6methylsalicylate. Heterologous expression of nataxazole biosynthesis cluster leads to the production of AJI9561.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Scheme 1

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Scheme 2

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Table of Contents

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