Regulation of daunorubicin biosynthesis in Streptomyces peucetius ...

Environment Health Techniques 636

Ajithkumar Vasanthakumar et al.

Review Regulation of daunorubicin biosynthesis in Streptomyces peucetius – feed forward and feedback transcriptional control Ajithkumar Vasanthakumar1, Karuppasamy Kattusamy2 and Ranjan Prasad2 1 2

Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Melbourne, Victoria, Australia Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India

Streptomyces are a major group of soil bacteria that produce wide range of bioactive compounds including antibiotics. Daunorubicin is a chemotherapeutic agent for treatment of certain types of cancer, which is produced as a secondary metabolite by S. peucetius. Owing to the significance of this drug in treating cancer, understanding the molecular mechanism of its biosynthesis will assist in the genetic manipulation of this strain for better drug yields. Additionally, the knowledge can also be applied to design hybrid antibiotics that can be made in vivo by transferring genes from one Streptomyces species to another. Biosynthesis of daunorubicin in S. peucetius is accomplished by the function of 30 enzyme-coding genes in a sequential and coordinated fashion. In addition to these enzymes, three transcriptional regulators DnrO, DnrN and DnrI regulate this multi-step process by forming a coherent feed forward loop regulatory circuit, consequently controlling the entire enzyme coding genes. Since daunorubicin is a DNA intercalating drug, maintaining an optimal intracellular drug concentration is pivotal to prevent self-toxicity. Commencement of daunorubicin biosynthesis also activates the feedback mechanisms mediated by the metabolite. At exceeding intracellular concentrations, daunorubicin intercalates into DNA sequences and impedes the binding of these transcription factors. This feedback repression is relieved by a group of self-resistance genes, which concurrently efflux the excess intracellular daunorubicin. This review will discuss the mechanistic role of each transcription factor and their interplay in initiating and maintaining the biosynthesis of daunorubicin in S. peucetius. Keywords: Streptomyces peucetius / Daunorubicin / Feedback regulation / Transcription / Coherent feed forward loop Received: June 3, 2012; accepted: August 3, 2012 DOI 10.1002/jobm.201200302

Introduction Soil bacteria belonging to the genus Streptomyces are key producers of several antibiotics and bioactive compounds, which are products of secondary metabolism. Polyketides are large group of secondary metabolites that include antibiotics like tetracycline and erythromycin. Polyketides are further classified into Type I, II and III compounds based on the enzymes involved in their biosynthesis. A notable member of Type II polyketide family is Daunorubicin (DNR), produced by the pigmented bacteria Streptomyces peucetius. DNR, a widely used Correspondence: Ranjan Prasad, Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India E-mail: [email protected] Phone: þ91 0452 2459115 Fax: þ91 0452-2459105 ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

anticancer drug and its hydroxylated derivative doxorubicin (DXR) [1] are synthesized by complex monofunctional enzymes collectively known as Type II polyketide synthases (PKS). However, the Type II PKS enzymes cannot complete the biosynthesis without the co-operation of several other enzymes that catalyze biochemical reactions like oxidation, hydroxylation, cyclation and glycosylation [2]. All these biosynthetic genes are clustered together to form a secondary metabolic island (SMILE) spanning a 40 kb region in the 8.7 Mb S. peucetius genome. Earlier studies focused on elucidating the biochemical process involved in the biosynthesis of DNR, primarily using mutants blocked at various stages of the biosynthetic pathway. Characterization of the 40 kb daunorubicin biosynthesis gene cluster [3, 4], that encompasses catalytic enzymes, transcription factors and resistance genes, enhanced the understanding of

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J. Basic Microbiol. 2013, 53, 636–644

Regulation of daunorubicin biosynthesis in Streptomyces peucetius

regulatory mechanisms that control DNR biosynthesis. By making use of dnrO, dnrN and dnrI transcription regulator mutants, the existence of tightly regulated antibiotic biosynthesis machinery was expounded. DNR being a DNA intercalating drug, self-resistance to this drug is essential for the survival of the organism. Selfresistance is achieved primarily by an ATP driven efflux pump DrrAB. Absence of this pump leads to down regulation of polyketide synthase genes, possibly due to feedback inhibition by DNR accumulated within the cell. In a wild type strain, such a feedback circuit accomplishes sustained biosynthesis of DNR. Such a feedback system is amenable to genetic manipulation for improving the yield of this antibiotic. This review will focus on how DNR biosynthesis and self-resistance is regulated by three transcription factors and the crucial role of the drug itself in maintaining homeostasis.

Daunorubicin biosynthesis – structural genes and the process The proposed DNR biosynthesis pathway (Fig. 1A) was constructed based on genetic and biochemical analysis of S. peucetius mutants blocked at different stages of the pathway. The partial genomic sequence data obtained from S. peucetius and its closest homolog Streptomyces sp strain C5 was compiled and comprehensive map of the 40 kb pathway gene cluster was derived. Biosynthesis of DNR starts with the formation of the first intermediate, a

Figure 1. Schematic diagram showing daunorubicin biosynthetic pathway (A) and the coherent feed forward loop network of the proximal transcriptional activators (B). During the later stages of S. peucetius growth, unknown signals initiate the DNR biosynthetic machinery by activating the first transcription factor DnrO. This transcription factor subsequently activates other transcription factors dnrN and dnrI. The master transcription factor DnrI activates all structural genes involved at various stages of DNR biosynthesis and also simultaneously activates the self-resistance genes drrA and drrB. ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

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21-carbon decaketide that happens by serial condensation of nine malonyl-CoA units to one propionyl-CoA starter unit. This multi-step reaction is sequentially catalyzed by enzymes of the polyketide synthase (PKS) family starting with the action of 3-oxoacyl: ACP synthase (dpsA) [5], ketosynthases (dpsB and dpsC) [6, 7], acyltransferase (dpsD) [7] and acyl carrier protein (dpsG) [8]. The decaketide then undergoes reduction by 9-ketoreductase (dpsE) [6], followed by intra-molecular aldol condensation and 1st ring cyclation catalyzed by DpsF [6]. The 2nd and 3rd ring cyclation is catalysed by DpsY to form 12deoxyalkalonic acid [9]. This intermediate is converted to alkalonic acid by mono-oyxgenase DnrG by the introduction of a keto group. Subsequently, alkalonic acid is converted to aklaviketone by alkalonic acid-S-adenosyl-Lmethionine methyl transferase, a homodimeric protein encoded by dauC/dnrC [3]. Another unusual protein alkalonic acid methyl ester cyclase encoded by dauD/ dnrD is also involved in the cyclation of alkalonic acid through a presumed intra-molecular aldol condensation [10, 11]. Finally, sequential action of the two enzymes aklaviketone reductase, which is encoded by dnrH and a hydroxylase encoded by dnrF reduces the 7-oxo moiety of aklaviketone to a hydroxy-group to form e-rhodomycinone [11]. In yet another series of biochemical reaction TDPdaunosamine is synthesized and this glycoside is attached to e-rhodomycinone. Further biochemical modifications produce DNR. The enzymes encoded by genes dnmL (thymidylyl transferase) [12], dauM (TDPglucose-4, 6-dehydratase) [13] initiates TDP-daunosamine biosynthesis by catalyzing the formation of the intermediate TDP-4-keto-6-deoxy-L-glucose. This intermediate is a known substrate for the enzyme dNDP-sugar 3,5 epimerase encoded by dnmU [14]. Subsequently, enzymes encoded by the genes dnmT [15], dnmQ [16] and dnmJ [4] sequentially catalyses the TDP-2, 6-deoxy-3-amino-4ketohexulose. The final step of TDP daunosamine biosynthesis is the reduction of TDP-2, 6-deoxy-3amino-4-ketohexulose to TDP-daunosamine by the dnmV gene product [14]. The aglycone moiety e-rhodomycinone lacking daunosamine sugar lacks biological activity. Hence glycosylation is a key step in the formation of biologically active final product. Glycosyl transferase enzymes encoded by genes dauH [17] and dnmS [16] catalyses the formation of rhodomycin D, the first glycosylated intermediate of the pathway. An esterase DnrP/DauP demethylates rhodomycin-D and converts it into 13-deoxycarminomycin [18]. This is followed by hydroxylation, oxidation and methylation of the former intermediate by DauK [18] to form 4-O-methyl rhodomycin-D. This intermediate undergoes C13 oxidation in

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two stages catalyzed by the cytochrome P450 enzyme encoded by the doxA gene, giving rise to DNR [8, 18, 19]. DNR is further oxidized to DXR, but this appears to be an incidental property of doxA protein, since C14 hydroxylation is 170 fold less efficient that C13 oxidation in vitro [19]. All these enzyme-coding genes have been shown to be regulated by the transcription factor DnrI [20] (Fig. 1A). However, the scheme of activation of all the genes is not known. It is also not clear if the pathway intermediates are involved in the activation of the gene that is involved in the catalysis of the subsequent reaction.

Self-resistance mechanisms Anthracycline family antibiotics function by intercalating to DNA. DNR belongs to the anthracycline family and exerts its function by DNA intercalation and subsequent inhibition of topoisomerase II. Because of its DNA intercalating nature, intracellular DNR threshold in S. peucetius is maintained below the lethal concentration using diverse mechanisms. Self-resistance to DNR in S. peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB [21] (Fig. 1A). The DrrA protein is similar to a large family of ATP-binding transport proteins, including the proteins encoded by the multidrug resistance genes (mdr) of mammalian tumor cells, which confer resistance to DNR, DXR and some other structurally unrelated chemotherapeutic agents [21]. DrrA is a peripheral membrane protein that binds ATP in a DXR-dependent manner and acts as energy transducing subunit [22]. DrrB is a hydrophobic integral membrane protein that functions as a transporter for the efflux of DNR and DXR [23]. The expression and function of DrrA and DrrB proteins were found to be dependent on each other. Together, DrrA and DrrB form an ATP-driven pump for the efflux of these drugs analogous to the functioning of MDR proteins in mammalian tumor cells. DrrB consists of eight transmembrane domains and a long N-terminal cytoplasmic domain, three cytoplasmic loops, and a short cytoplasmic C-terminal tail, which may form the potential sites of interaction with DrrA [24]. A mutant strain that lacks these resistance genes exhibits increased sensitivity to the drug. Besides, DNR production also declines to 10%. Overexpression of drrAB increased DNR production and self-resistance reinforcing the fact that apart from conferring resistance, these two resistance genes also play a crucial role in regulating the biosynthetic machinery [25]. Another self-resistance gene that functions very differently is the DrrC protein. This protein bears strong ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins that are involved in excision repair of DNA [26]. Since introduction of drrC into S. lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. Expression of drrC in an E. coli uvrA– strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of an uvrAþ strain. DrrC protein was demonstrated to bind to DNA only in the presence of ATP and DNR exhibiting a novel mechanism to confer self-resistance. It is believed that DrrC binds to DNR intercalated DNA sequences and dislodges the drug in an ATP dependent fashion. This resistance gene is induced by the metabolite DNR and is regulated by DnrN and DnrI [27].

Transcriptional regulators DNR biosynthesis in S. peucetius is regulated by three transcriptional regulators DnrO, DnrN and DnrI, which play a non-redundant and indispensable role in activating the biosynthetic machinery (Figs. 1 and 2). These genes

Figure 2. Daunorubicin biosynthesis gene regulatory network. DnrO is the first transcription factor activated in the DNR biosynthesis gene cluster. DnrO binds to promoter of the second transcription factor dnrN and activates it. The binding of DnrO to dnrN promoter is also an autoregulatory module for dnrO. The upstream transcription factors DnrO and DnrN activate the master regulator dnrI. This process can be either co-operative (DnrO and DnrN) or can be executed by DnrN alone. The master regulator DnrI activates all structural and selfresistance genes of the biosynthesis cluster and also activates a transcriptional repressor dnrW that can inhibit dnrI. Along with DnrW, the metabolite DNR it-self re-programs the transcriptional pathway by inhibiting each of the up-stream transcription factors by intercalating to their targets.

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are located within the biosynthetic gene cluster initially identified as DNA binding proteins by sequence similarity searches and functional analysis of mutations that affected DNR biosynthesis. A complete blockade of DNR biosynthesis in these mutants and the absence of any intermediates of the DNR pathway also confirmed that these genes function early during biosynthesis or prior to the activation of genes involved in the biosynthesis. Promoter reporter assays revealed transcriptional activity of these three genes as early as 24 h prior to the commencement of DNR biosynthesis. DnrI showed high degree of similarity with actII orf4 of S. coelicolor and AfsR [28]. The role of dnrI as a transcriptional activator was confirmed by the increase in actinorhodin production when extra copies of dnrI were introduced in S. lividans [20]. Similarly dnrI also complemented actII orf4 mutation in a heterologous background. Transcript analysis of drrAB, dnrZUV, dnrPKS and dpsABC operons and the drrC gene in dnrI mutant background showed that DnrI functions as transcription factor that activates these genes thereby key biosynthetic reactions of the pathway are activated in this manner [4]. In addition, it was demonstrated that DnrI binds to intergenic region of dnrG-dpsE and dnrC-dnrD [20]. Multiple target activation is a property of DnrI (master transcription factor) and it has a dominant role in driving DNR biosynthesis. The second activator protein DnrN was initially thought to be a response regulator that controlled DNR biosynthesis utilizing a two-component signaling module [29]. DnrN sequence was compared with other response regulators and an aspartate residue (D55) was predicted to be involved in the phosphorylation reaction. To ascertain the role of D55, this conserved residue was substituted with glutamate (E) and asparagine (N). These changes did not affect DNR biosynthesis disproving the notion that DNR biosynthesis is controlled by two-component signal transduction pathway that

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involved DnrN [29]. DnrN gene is divergently transcribed from the adjacent gene dnrO and is separated by a 433 bp intergenic region that has promoters of both regulatory genes [30]. DnrO was originally thought to be a repressor due to its sequence similarity to genes like Tetracinomycin C resistance gene repressor (TcmR) of S. glaucescens, biotin operon repressor (BirA) of E. coli and tetracycline resistance gene repressor (TetR). Contrary to the predictions, inactivation of dnrO in S. peucetius abolished DNR biosynthesis [31]. DnrO is the first gene activated in the pathway that has three promoters Op1, Op2 and Op3 with the transcripts starting from 4, 317 and 386 bp upstream of the start codon of dnrO [31] (Fig. 3). Transcriptional start for dnrN was also identified as 319 bp upstream of its predicted ATG start codon [31] (Fig. 3). At least two transcripts of dnrO overlap with opposing dnrN transcript.

Transcriptional repressor drrD/dnrW In addition to the transcriptional activators, recently a gene that was previously known as a self-resistance gene from the sequence homology analysis was characterized and reported as the first repressor that regulates the pathway. This gene drrD, also known as dnrW is located between the drrB and dnrX genes in the DNR cluster and based on conserved domain analysis was presumed to be of the FAD dependant oxido-reductase family. Recently Yuan et al. [32] inactivated this gene in yet another DNR producer S. coeruleobidus and established its role in repressing the master transcription factor dnrI (Fig. 2). Deletion of dauW (drrD and dnrW ortholog in S. coeruleobidus) resulted in the increase of dnrI transcripts, which subsequently increased DNR biosynthesis by 8 fold. Since DnrI is a known transcriptional activator of drrAB DNR efflux genes, increase

Figure 3. Intergenic region of dnrN and dnrO genes with alternative promoters and opposing transcripts. The genes coding for transcriptional activators DnrO and DnrN are arranged and transcribed divergently. Furthermore, the dnrO gene also features three promoters, each coding transcripts with varying length 50 -UTRs. These UTRs overlap with the 50 -UTR of the dnrN gene. ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

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in DnrI levels also enhanced self-resistance to DNR in a dauW mutant background [32]. However evidence for activation of drrAB operon by DnrI is lacking.

this case where, DnrO activates dnrN and both DnrO and DnrN activate dnrI for downstream activation of other genes of the pathway (Figs. 1 and 2).

Transcriptional control by a coherent feed forward loop

Feed back regulation

Gene regulation by coherent feed forward loop (CFFL) involves two transcription factors X and Y amplifying the final gene target Z. These network motifs has been extensively studied in E. coli systems [33] and several variants of CFFL like SUM-FFL has also been described before in E. coli [34]. In S. peucetius, the three transcriptional regulators act in tandem to form a feed-forward loop to initiate the biosynthesis of DNR (Figs. 1 and 2). The stepwise activation of these genes to form this regulatory loop was unraveled with the help of S. peucetius mutants for each of the transcription factors and complementation assays. This feed forward loop could be an amplification step in response to extracellular signals for attaining optimum transcriptional output. Although the exact signals that activate dnrO has not been identified, this is the first gene known to be activated in the pathway [30, 31]. The position of dnrO on the top of the activation hierarchy was confirmed by complementing dnrO null mutant using multi-copy dnrN and dnrI genes [31]. Complementation of this DNR defective mutant using the two transcriptional regulators in isolation and in combination rescued the defect validating that dnrO acts proximally to either dnrN or dnrI [31]. Binding of DnrO to the overlapping dnrN promoterdnrOp1 operator sequence was later reported to exhibit two different outputs: activation of dnrN and autoregulation of dnrO itself [35]. DnrN in turn activates the master transcription factor dnrI. This was confirmed when dnrI transcript was not found in dnrN null mutant [29]. Additionally, DNA binding studies using gel mobility shift assay demonstrated the sequence specific binding of DnrN to upstream region of dnrI [29]. . DNR biosynthesis resumed when the dnrN null mutant was complemented with dnrI under promoters like ermE and tipA [30]. Interestingly, when dnrN was expressed in multi copies, it did not influence the expression of dnrI [29]. The dependence of dnrO for dnrI expression was also demonstrated by fusing melC to dnrIp and analyzing its expression in the presence of either dnrN or dnrO or both in a heterologous host S. lividans. The strain which carried both dnrN and dnrO genes showed higher expression of melanin indicating enhanced activity of dnrI promoter [35]. This finding confirms that a coherent feed forward loop (CFFL) transcriptional module exists in ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

Many DNA intercalating drugs appear to function by interfering with critical cellular processes such as inhibition of DNA and RNA polymerase, topoisomerases and nucleases [36, 37]. Mechanistically these drugs block the progress of processive enzyme complexes or interfere with protein access to DNA. The organisms, which synthesize these DNA binding drugs, adopt various mechanisms to escape the cytotoxic effects. Mechanisms by which organism protects itself from antibiotic toxicity is by drug inactivation, target site modification, reduction of intracellular concentration via efflux and drug binding [38]. Bleomycin, which is a DNA alkylating agent, is modified for self-resistance by Bat (Bleomycin Nacetyltransferase) and binding by BLMA (bleomycin (Bm)binding protein proteins in S. verticillius [39]. S. lavendulae produces mitomycin C which is also a DNA damaging agent. Self-resistance in this organism is by a flavoprotein that reoxidizes the activated mitomycin C [40]. S. peucetius exploits the DNA intercalating property of DNR to modulate the expression of the transcription factors to shut down the biosynthesis machinery. In addition to the DrrAB efflux pump and DrrC encoded self-resistance; S. peucetius also utilizes a feedback mechanism to repress the expression of the transcriptional activators that inhibit the biosynthesis of drug in the first place. Since DnrN has a crucial role in activating the master regulator dnrI, a small molecule screen was performed to identify inhibitors that affect the binding of DnrN to the dnrI promoter sequence. Among several small molecules tested, only DNR inhibited this interaction and this was due to DNR intercalating to DNA [29]. The presence of preferential DNR intercalating site, a GC pair flanked by A/T in the DnrN binding sequence also confirmed this. Intercalation of DNR to DNA will alter the topology of the double stranded structure and create steric hindrance for DnrN to bind. This in vitro observation can be extrapolated to an in vivo situation wherein excess intracellular DNR presumptively inhibits transcriptional activation of dnrI [29]. This mode of inhibition may act as a negative feed back regulatory mechanism to regulate the biosynthesis of DNR when it exceeds its optimal physiological concentration. However, no in vivo experiments have been performed to validate this phenomenon with regards to DnrN and dnrI. On the other hand extensive work has been done to confirm

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Table 1. Structural and regulatory genes that are involved in DNR biosynthesis. Gene names Functions known

References

dnmL dnmM dnrN dnrO dnrF drrA drrB drrD/dnrW dnrX dpsY dnmZ dnmU dnmV dnmJ dnrI doxA dnrV dnrU dpsG dpsH dnmT dnrH dnrE dpsF dpsE dnrG dpsA dpsB dpsC dpsD dnrC dnrD dnrK dnrP dnmQ dnmS drrC

[12] [12] [29] [31] [44] [22, 25] [22, 25] [2] [9] [9] [14] [14] [14] [4] [20] [18] [8] [8] [6] [45] [15] [15] [17] [6] [6] [45] [5] [6] [46] [7] [4] [4] [18] [18] [16] [16] [26, 27]

TDP-glucose thymidylyl transferase that generates dTDP-glucose TDP-glucose 4,6-dehydratase; non-functional gene in S.peucetius Pseudo response regulator/transcriptional activator Transcriptional activator/autoregulator Aklavinone 11-hydroxylase ATP-binding protein that associates with DrrB for DNR eff lux Membrane bound hydrophobic protein involved in DNR eff lux Transcriptional repressor of dnrI Metabolism of daunorubicin to acid sensitive compounds Synthesis of 12-deoxyaklanonic acid Flavoprotein acting at some step in daunosamine biosynthesis instead of an acyl-CoA dehydrogenase TDP-4-keto-6-deoxyglocose 3,5-epimerase TDP-4-keto-6-deoxyglocose 3,5-hexulose Addition of N-function to TDP hexulose Key transcriptional regulator Daunorubicin 14-hydroxylase, 13-deoxyanthracycline hydroxylase Unknown Ketoreductase specific for the C-13 carbonyl of daunorubicin Acyl carrier protein for polyketide synthase Unknown; possible role in polyketide assembly Unknown function in TDP daunosamine biosynthesis Glycosyl transferase Aklavinone reductase Polyketide cyclase involved in aklanoic acid formation Polyketide reductase 12-Deoxyaklanoic acid oxygenase Polyketide synthase:Keto acyl synthase-a subunit (Possess active-SH) Polyketide synthase:Ketoacyl synthase-b subunit Homolog of KAS-III but lacking active site; might assist in propionyl-SCoA starter units election Acyl-SCoA:ACP acyltransferase; assist in specifying propionyl-SCoA starter unit selection Aklanoic acid methyltransferase Aklanoic acid methyl ester cyclase Anthrocycline 4-O-methyl transferase Rhodomycin D 16 methylesterase Involved in unknown reaction in TDP-daunosamine biosynthesis TDP-daunosamine; -rhodomycinone glycosyl transferase. Resistance gene involved in the removal of intercalated DNR from DNA

the existence of a feed back mechanism at the DnrO-dnrN node. Similar to the DNR inhibition observed with DnrN-dnrI promoter interaction, DnrO-dnrN promoter interaction was also restrained by the metabolite [35]. This observation was further confirmed by an in vivo experiment in which dnrO full-length gene was fused to a reporter melC and in the presence of DNR the expression of melC increased. Unlike the former setting, this inhibition not only inhibits the activation of dnrN but also relieves the auto regulation of dnrO itself. When a truncated version of DnrO was used, DNR did not have any effect on the expression of the reporter gene [35]. Based on this Jiang and Hutchinson [35] proposed a model in which they claimed that DnrO has two binding sites, one overlapping the dnrO promoter and the second upstream of dnrN promoter. In the presence of DNR, the DnrO ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

contact overlapping its own promoter will be inhibited retaining the contact upstream of dnrN. In contrast to the DNR-DNA mechanism, the authors hypothesized DNRinteracting to DnrO protein itself in this case. Our study however demonstrated the same phenomenon using EGFP fused to dnrN and dnrO promoter and analysis of the expression in the presence of full-length dnrO and DNR in a heterologous host S. lividans [41]. We found a decrease in dnrN expression in the presence of DNR and an inverse in dnrO expression. We also calculated the minimum inhibitory concentration of DNR needed to execute this negative feed back control [41]. In agreement to the increase in dnrO levels in the presence of excess DNR, the drrAB mutant created in our lab also illustrated an increase in the dnrO transcript levels when quantified by PCR. The drrAB mutant has low selfresistance due to accumulation of DNR [25]. All these

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observations together confirm the existence of DNR dictated feed back mechanism in S. peucetius that acts at different nodes to ensure modulation of the biosynthesis machinery (Fig. 2).

Future perspectives Although much progress has been made in understanding the regulatory genes and the mechanisms employed by these genes to control DNR biosynthesis, many questions remain unanswered. One important question is the nature of the extracellular signal that induces the biosynthesis pathway. In majority of the Streptomyces species, antibiotics or secondary metabolites synthesis are induced by small quorum sensing molecules called g-butyrolactones. These small molecules act as extracellular signals, which will be recognized by two component signal transduction modules to commence biosynthesis of metabolites. However in S. peucetius, a small molecule inducer or two component signaling modules have not been identified. Whole genome sequencing completed by Parajuli et al. [42] might shed light into the existence of any two-component systems possibly involved in DNR biosynthesis. In an attempt to purify proteins that bind to the intergenic region between dnrN and dnrO genes, we purified and identified a DNA binding protein that possessed a dehydrogenase domain [43]. However, this study did not reveal the significance of this binding. The inter-genic region between dnrN and dnrO genes is packed with regulatory motifs, and this region might be crucial for sensing the unknown extracellular signal. Presence of three promoters for dnrO gene also suggests that these three promoters can respond to different activation signals or can recruit different sigma factors. Another possibility is that the three promoters can be activated at different time points during the vegetative growth and therefore might regulate dnrO temporally. DnrO executes dual function namely, activation of dnrN and auto-regulation. Two proximal promoters of dnrO are active during DNR biosynthesis. The two transcripts from these promoters have a long 50 -UTR that overlap with the 50 UTR of dnrN gene. Knowing that these genes should be tightly regulated to prevent self-toxicity, additional regulatory mechanisms that regulate the expression of the regulatory genes might exist. We anticipate that the 50 -UTR’s from the two divergent genes could form RNA-RNA hybrid, which may lead to transcriptional interference. Transcriptional collision is a common event that can impede transcription of genes with opposing promoters. In addition to transcriptional interference by RNA hybrid ß 2013 WILEY-VCH Verlag GmbH Co. KGaA,Weinheim

formation, transcriptional collision of RNA polymerases that drive the two proximal promoters of dnrO and the dnrN promoter is a possibility. Coherent feed forward loop serves as a signal amplification step during transcriptional activation. In the case of S.peucetius although DnrO activates dnrN and both together activate dnrI, which in turn activates other genes, a quantitative transcript analysis is lacking. Furthermore, additional experimental data is also required to prove the cooperative role of DnrO and DnrN in activating the master regulator dnrI. The study so far on DNR biosynthesis is limited to the 40 kb gene cluster due to the lack of whole genome sequencing data. Even though a near complete picture of the daunorubicin biosynthesis pathway has been elucidated from the known genes, the possibility of genes external to the cluster having a vital role cannot be discounted. With the S. peucetius genome completely sequenced, its feasible to revisit the DNR biosynthesis pathway that may re-define it in the future.

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dnrX genes govern early and late steps of daunorubicin and doxorubicin biosynthesis. J. Bacteriol., 180, 2379–2386.

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