Implication of orphan histidine kinase (OhkAsp) in biosynthesis of doxorubicin and daunorubicin in Streptomyces peucetius ATCC 27952.

The orphan histidine kinase (HK) from Streptomyces peucetius ATCC 27952 (ohkAsp) was found to be implicated in the regulation of doxorubicin (DOX)/daunorubicin (DNR) biosynthesis, self-defense and developmental attributes. OhkAsp is a homolog of OhkA from Streptomyces coelicolor and Streptomyces avermitilis (with 73 and 75% identity). As in its homologs, S. peucetius mutant with deletion of ohkAsp was found to enhance metabolite biosynthesis and impaired the morphological differentiation. But, unlike its homologs from Streptomyces coelicolor and Streptomyces avermitilis, differential enhancement in level of secondary metabolite production was found in overexpression mutants apart from deletion mutant. The deflection in characteristics of OhkA in its homologue from S. peucetius ATCC 27952, and its imminent implications was monitered by making various mutants with differential expression level of ohkAsp. The variations were observed in the morphology of mutants, transcriptional level of effectors and regulators of DOX/DNR biosynthesis pathway, DOX/DNR precursor pool and biomass accumulation. Based on comparisons of domain arrangements among its homologs, Low Complexity Region (LCR) present on the OhkAsp was the only domain that stood out. Further, the LCR on OhkAsp was found to be overlapping with a putative receiver domain responsible for interaction with response regulator. The imminent implications of differential expression level of ohkAsp on: regulation and biosynthesis of DOX/DNR, morphological differentiation, DOX/DNR precursor pool and biomass accumulation were explored in this study.

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.

Hydroxylation of Resveratrol with DoxA In Vitro: An Enzyme with the Potential for the Bioconversion of a Bioactive Stilbene.

The late-stage doxorubicin biosynthesis pathway acting enzyme (DoxA) from Streptomyces peucetius CYP129A2 exhibited substrate promiscuity towards the stilbene group of compounds such as resveratrol. DoxA along with two accessory enzymes ferrdoxin reductase and ferredoxin from spinach hydroxylated resveratrol at the 3'-position in vitro to produce piceatannol. The product was identified by HPLC-PDA and high-resolution HR-qTOF-ESI/MS analyses in positive mode. The ESI/MS fragments resembled the hydroxylated product of resveratrol.

Complete genome sequence of Streptomyces peucetius ATCC 27952, the producer of anticancer anthracyclines and diverse secondary metabolites.

Streptomyces peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. peucetius ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain.

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL-1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10-4) when compared to other reports from this genus (1.1×10-5 to 2.5×10-8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.

Correlation of in Situ Oxazolidine Formation with Highly Synergistic Cytotoxicity and DNA Cross-Linking in Cancer Cells from Combinations of Doxorubicin and Formaldehyde.

Anthracyclines are a class of antitumor compounds that are successful and widely used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA cross-linker. Contrary to current dogma, we show that both efficient DNA cross-linking and potent synergy in combination with formaldehyde correlate with the anthracycline's ability to form cyclic formaldehyde conjugates as oxazolidine moieties and that the cyclic conjugates are better cross-linking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development.

Tissue retention of doxorubicin and its effects on cardiac, smooth, and skeletal muscle function

Cancer-related fatigue is a pervasive syndrome experienced by a majority of cancer patients undergoing treatment, and muscular dysfunction may be a key component in the development and progression of this syndrome. Doxorubicin (DOX) is a commonly used antineoplastic agent used in the treatment of many cancers. The purpose of this study was to determine the effect of DOX exposure on the function of cardiac, skeletal, and smooth muscle tissues and examine the role accumulation of DOX may play in this process. In these studies, rats were treated with DOX and measurements of cardiac, skeletal, and smooth muscle function were assessed 1, 3, and 5 days after exposure. All muscular tissues showed significant and severe dysfunction, yet there was heterogeneity both in the time course of dysfunction and in the accumulation of DOX. Cardiac and skeletal muscle exhibited a time-dependent progressive decline in function during the 5 days following DOX treatment. In contrast, vascular function showed a decline in function that could be characterized as rapid onset and was sustained for the duration of the 5-day observation period. DOX accumulation was greatest in cardiac tissue, yet all muscular tissues showed a similar degree of dysfunction. Our data suggest that in muscular tissues both DOX-dependent and DOX-independent mechanisms may be involved with the muscular dysfunction observed following DOX treatment. Furthermore, this study highlights the fact that dysfunction of skeletal and smooth muscle may be an underappreciated aspect of DOX toxicity and may be a key component of cancer-related fatigue in these patients (AU)

Transgenic CCL2 expression in the central nervous system results in a dysregulated immune response and enhanced lethality after coronavirus infection.

Chemokine (C-C motif) ligand 2 (CCL2), a chemoattractant for macrophages, T cells, and cells expressing CCR2, is upregulated during acute and chronic inflammation. CCL2 has been implicated in both proinflammatory and anti-inflammatory responses and has been suggested as a target for therapy in some inflammatory disorders. To examine the role of CCL2 during virus infection, we infected mice transgenically expressing CCL2 in the central nervous system (CCL2 Tg) with an attenuated neurotropic coronavirus (rJ2.2 strain of mouse hepatitis virus). Infection of wild-type mice with rJ2.2 results in mild acute encephalitis, followed by a nonlethal, chronic demyelinating disease. Proinflammatory innate and adaptive immune responses mediate virus clearance. In marked contrast, CCL2 Tg mice infected with rJ2.2 ineffectively cleared virus and rapidly succumbed to the infection. CCL2 Tg mice mounted a dysregulated immune response, characterized by augmented accumulation of regulatory Foxp3(+)CD4(+) T cells and of nitric-oxide- and YM-1-expressing macrophages and microglia, suggestive of mixed M1/M2 macrophage activation. Further, macrophages from infected CCL2 Tg brains relative to non-Tg controls were less activated/mature, expressing lower levels of major histocompatibility complex class II (MHC-II), CD86, and CD40. Collectively, these results show that persistent CCL2 overexpression establishes and sustains an immunological milieu that is both inflammatory and immunosuppressive and predisposes mice to a defective immune response to a minimally lethal virus.

Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952.

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.

Biotechnological doxorubicin production: pathway and regulation engineering of strains for enhanced production.

Doxorubicin (DXR) is an anthracycline-type polyketide, typically produced by Streptomyces peucetius ATCC 27952. Like the biosynthesis of other secondary metabolites in Streptomyces species, DXR biosynthesis is tightly regulated, and a very low level of DXR production is maintained in the wild-type strain. Despite that DXR is one of the most broadly used and clinically important anticancer drugs, a traditional strain improvement strategy has long been practiced via recursive random mutagenesis, with little understanding of the molecular genetic basis underlying such enhanced DXR production. Since DXR titer enhancement is imperative in the fermentation industry, attaining a comprehensive understanding and its application of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this mini-review, various efforts to improve the titers of DXR have been summarized based on biosynthetic and regulatory studies including transcriptional and product analyses.

Limitations in doxorubicin production from Streptomyces peucetius.

Doxorubicin (DXR), produced by Streptomyces peucetius ATCC 27952, exhibits potent antitumor activity against various cancer cell lines. Considerable time has lapsed since the biosynthesis of DXR and its overproduction was first summarized. Based on biosynthetic studies and product analysis, various factors affecting its production by the parental strain, S. peucetius ATCC 27952, are reviewed to better circumvent any bottlenecks in DXR production, thereby providing ideas to genetically engineered industrial strains of S. peucetius.

Identification of the duplicated genes for S-adenosyl-l-methionine synthetase (metK1-sp and metK2-sp) in Streptomyces peucetius var. caesius ATCC 27952.

AIMS: To characterize the function of both metK1-sp (sp1190) and metK2-sp (sp1566) in vitro and in vivo, and to study the regulation of doxorubicin production by overexpressing the metK. METHODS AND RESULTS: We cloned two orfs into pET32a(+) respectively, and the formation of S-Adenosyl-l-methionine was clearly observed in the in vitro enzyme assays as functional MetKs. Reverse transcriptase polymerase chain reaction (PCR) analysis indicated that the transcripts for the metK1-sp were repressed as Streptomyces cells entered the decline phase, whereas that of the metK2-sp was induced, suggesting that these MetK proteins may be important for the growth and the regulation of secondary metabolites during the stationary growth phase, whether considered together or separately. Furthermore, we found that the introduction of high-copy-number plasmids containing the metK1-sp and metK2-sp resulted in 2.1- and 1.4-fold greater levels of doxorubicin production than the control transformants containing only the vector, respectively. We also attempted to disrupt the metK-sp and found that doxorubicin production from the metK1-sp-deleted mutant (Streptomyces peucetius/pNN1) was reduced when compared to the parent strain (S. peucetius var. caesius ATCC 27952). CONCLUSIONS: The results of this study indicated that two metK are differentially expressed during cell growth, and that the expressions of the two metK genes are differentially regulated under the same conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces peucetius var. caesius contains two genes, metK1-sp and metK2-sp, which encode functional S-adenosyl-l-methionine synthetase (MetK). The degree of homology (90% identity) found between the two genes shows that metK1-sp and metK2-sp are duplicated genes. Although there is currently no evidence for the relationship of the duplicated metK genes involved in the regulation of doxorubicin production, metK1-sp and metK2-sp may play a role in controlling the stimulation of antibiotic production during secondary metabolism.

Improvement in doxorubicin productivity by overexpression of regulatory genes in Streptomyces peucetius.

Biosynthesis of doxorubicin (DXR) is tightly regulated, limiting its production in Streptomyces peucetius cultures. The regulatory genes dnrN, dnrI, afsR, and metK1-sp from S. peucetius ATCC 27952 were cloned into the pIBR25 expression vector under the control of the strong ermE* promoter to enhance DXR production. The constructed expression plasmids, pNI25 (with dnrN-dnrI), pNIS25 (with dnrN-dnrI-metK1-sp), pNIR25 (with dnrN-dnrI-afsR), pRS25 (with afsR-metK1-sp) and pNIRS25 (with dnrN-dnrI-afsR-metK1-sp), were transformed into S. peucetius. The recombinant strains NI, NIS and NIR produced greater amounts of DXR than the parental strain with an increment of 1.2-fold by pNI25, 1.4-fold by pNIS25 and 4.3-fold by pNIR25, whereas pRS25 and pNIRS25 had no significant effect on DXR production. We also studied the transcriptional level of overexpressed regulatory genes and relative production of DXR, daunorubicin (DNR) and epsilon-rhodomycinone (RHO) in each recombinant strain.

Self-resistance mechanism in Streptomyces peucetius: overexpression of drrA, drrB and drrC for doxorubicin enhancement.

The resistance genes drrABC from Streptomyces peucetius ATCC 27952 were cloned into the pIBR25 expression vector under a strong ermE* promoter to enhance doxorubicin (DXR) production. The recombinant expression plasmids, pDrrAB25, pDrrC25 and pDrrABC25, were constructed to overexpress drrAB, drrC and drrABC, respectively, in S. peucetius ATCC 27952. The recombinant strains produced more DXR than the parental strain: a 2.2-fold increase with pDrrAB25, a 5.1-fold increase with pDrrC25, and a 2.4-fold increase with pDrrABC25. We also studied the relative ratios of doxorubicin, daunorubicin and epsilon-rhodomycinone produced in these recombinant strains.

Isolation and genetic manipulation of the antibiotic down-regulatory gene, wblA ortholog for doxorubicin-producing Streptomyces strain improvement.

Cross-genome comparative transcriptome analyses were previously conducted using the sequenced Streptomyces coelicolor genome microarrays to understand the genetic nature of doxorubicin (DXR) and daunorubicin (DNR) overproducing industrial mutant (OIM) of Streptomyces peucetius. In this previous work, a whiB-like putative transcription factor (wblA ( sco )) was identified as a global antibiotic down-regulator in S. coelicolor (Kang et al., J Bacteriol 189:4315-4319, 2007). In this study, a total genomic DNA library of a DXR/DNR-overproducing S. peucetius OIM was constructed and screened using wblA ( sco ) as a probe, resulting in the isolation of a wblA ortholog (wblA ( spe )) that had 95% amino acid identity to wblA ( sco ). Gene disruption of wblA ( spe ) from the S. peucetius OIM resulted in an approximately 70% increase in DXR/DNR productivity, implying that the DXR/DNR production in the S. peucetius OIM could be further improved via comparative transcriptomics-guided target gene manipulation. Furthermore, several putative wblA ( spe ) -dependent genes were also identified using S. coelicolor interspecies DNA microarray analysis between the S. peucetius OIM and wblA ( spe )-disrupted S. peucetius OIM. Among the genes whose expressions were significantly stimulated in the absence of wblA ( spe ), the overexpression of a conserved hypothetical protein (SCO4967) further stimulated the total production of DXR/DNR/akavinone by 1.3-fold in the wblA ( spe )-disrupted S. peucetius OIM, implying that the sequential genetic manipulation of target genes identified from interspecies comparative microarray analysis could provide an efficient and rational strategy for Streptomyces strain improvement.

Precursor for biosynthesis of sugar moiety of doxorubicin depends on rhamnose biosynthetic pathway in Streptomyces peucetius ATCC 27952.

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 contains a TDP-D-glucose 4,6-dehydratase gene, dnmM, that is putatively involved in the biosynthesis of daunosamine, but the gene contains a frameshift in the DNA sequence that would cause premature termination of translation. In pursuit of another TDP-D-glucose 4,6-dehydratase in S. peucetius, a homologue gene, rmbB, was found, whose deduced product exhibits high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was located within a putative rhamnose biosynthetic gene cluster at another locus in the genome. RmbB was verified to be a functional TDP-D-glucose 4,6-dehydratase by enzyme assay as it catalyzed the conversion of TDP-D-glucose into TDP-4-keto-6-deoxy-D-glucose. Inactivation of rmbB in the S. peucetius genome abolished the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restored the doxorubicin production. Hence, rmbB provides TDP-4-keto-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin.

Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

Reversible derivatization to enhance enzymatic synthesis: chemoenzymatic synthesis of doxorubicin-14-O-esters.

An efficient three-step, chemoenzymatic synthesis of unprotected doxorubicin-14-O-esters from doxorubicin hydrochloride salt is described. The key step is a lipase-catalyzed regioselective transesterification/esterification using commercially available acyl donors and doxorubicin reversibly derivatized with N-alloc to improve substrate loadings. The overall yield is ca. 60% and chromatographic purification is not required, thereby making the process more amenable to scale-up.

Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell death.

Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.