A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of -rhodomycinone to rhodomycin D

Background: The biological activity of many microbial products requires the presence of one or more deoxysugar molecules attached to agylcone. This is especially prevalent among polyketides and is an important reason that the antitumor anthracycline antibiotics are avid DNA-binding drugs. The ability to make different deoxyaminosugars and attach them to the same or different aglycones in vivo would facilitate the synthesis of new anthracyclines and the quest for antitumor drugs. This is feasible using the numerous bacterial genes for deoxysugar biosynthesis that are now available. Results: Production of thymidine diphospho (TDP)- -daunosamine (dnm), the aminodeoxysugar present in the anthracycline antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), and its attachment to -rhodomycinone to generate rhodomycin D has been achieved by bioconversion with a strain of Streptomyces lividans that bears two plasmids. One contained the Streptomyces peucetius dnmJVUZTQS genes plus dnmW (previously named dpsH and considered to be a polyketide cyclase gene), dnrH, which is not required for the formation of rhodomycin D, and dnrl, a regulatory gene required for expression of the dnm and drr genes. The other plasmid had genes encoding glucose-1-phosphate thymidylyltransferase and TDP-glucose-4,6-dehydratase (dnmL and dnmM, respectively, or mtmDE, their homologs from Streptomyces agrillaceus) plus the drrAB DNR/DXR resistance genes. Conclusions: The high-yielding glycosylation of the aromatic polyketide -rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for -daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.     

The neocarzinostatin biosynthetic gene cluster from Streptomyces carzinostaticus ATCC 15944 involving two iterative type I polyketide synthases

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.     

Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.     

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.     

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.     

Hybrid anthracyclines from a genetically engineered Streptomyces galilaeus mutant

The genetic engineering of antibiotic-producing Streptomyces strains is an approach that is emerging and ready to become established as a successful methodology in developing analogues of the original, pharmaceutically important, natural products obtained from the organisms. The current report highlights this succes by demonstrating the high-level production of novel anthracyclines. The biosynthetic pathways of the nogalamycin-producing Streptomyces nogalater and the aclacinomycin-producing S. galilaeus were combined by transferring the genes of S. nogalater polyketide synthetase into a nonproducing S. galilaeus mutant. The resulting anthracycline antibiotics that were produced possessed structural features characteristic of compounds from both of the undoctored Streptomycesstrains.     

Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans.

BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.     

Characterization of the alnumycin gene cluster reveals unusual gene products for pyran ring formation and dioxan biosynthesis

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.     

Expanding the scope of aromatic polyketides by combinatorial biosynthesis

Combinatorial biosynthesis is a technology for mixing genes responsible for the biosynthesis of secondary metabolites, in order to generate products for compound libraries serendipitously or to cause desired modifications to natural products. Both of these approaches are extremely useful in drug discovery. Streptomyces and related species are abundant in bioactive secondary metabolites and were therefore the first microbes to be used for combinatorial biosynthesis. Polyketides are the most abundant medicinal agents among natural products. Structural diversity and a wide scope of bioactivities are typical of the group. However, the common feature of polyketides is a biosynthetic process from simple carboxylic acid residues. In molecular genetics, polyketides are sub-classified as types I and II, called modular and aromatic polyketides respectively. The best-known bioactivities of aromatic polyketides are their antibacterial and antitumor effects. Genetic analysis of aromatic polyketides has resulted in almost 30 cloned and identified biosynthetic gene clusters. Several biosynthetic enzymes are flexible enough to allow their use in combinatorial biosynthesis to create high diversity compound libraries. This review describes the state of the art of combinatorial biosynthesis, giving anthracyclines as examples. Contiguous DNA sequences for antibiotics, cloned from four different anthracycline producers, provide tools for rapid lead optimization or other structural modification processes, and not only for anthracyclines. Two gene cassettes enabling fast and flexible structural modification of polyketides are introduced in this paper.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate ‘Streptomyces maritimus’: evidence for the derailment of an aromatic polyketide synthase

Background: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-β-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain ‘Streptomyces maritimus’ deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides.Results: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from ‘S. maritimus’ has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis.Conclusions: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Characterization of the maduropeptin biosynthetic gene cluster from Actinomadura madurae ATCC 39144 supporting a unifying paradigm for enediyne biosynthesis

The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apoprotein was isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs--the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-alpha-tyrosine; a unique type of enediyne apoprotein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by combinatorial biosynthesis and establish a unified paradigm for the biosynthesis of enediyne polyketides.     

Genome-inspired search for new antibiotics. Isolation and structure determination of new 28-membered polyketide macrolactones, halstoctacosanolides A and B, from Streptomyces halstedii HC34

During the search for polyketide synthase (PKS) in the genome of Streptomyces halstedii HC34, we found clustered new genes which appeared to encode typical Type 1 PKSs beyond the cluster harboring the genes for the biosynthesis of antitumor antibiotic vicenistatin. The deduced domain configuration of these putative PKS genes allowed to predict a corresponding partial structure of polyketide, which was in turn materialized by isolation of new polyketide macrolactone halstoctacosanolides A and B from the fermentation broth of S. halstedii HC34. The structures of these metabolites were determined by spectroscopic means to have a novel 28-membered macrolactone structure. The partial structure deduced from the genetic data was completely compatible to the structures of halstoctacosanolides A and B. This success clearly demonstrates the present new approach of genome-inspired search for new antibiotics promising. Halstoctacosanolides A and B showed moderate antimicrobial activity against several microorganisms.     

Biosynthesis of the antitumor agent chartreusin involves the oxidative rearrangement of an anthracyclic polyketide

Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.     

The hedamycin locus implicates a novel aromatic PKS priming mechanism

The biosynthetic gene cluster for the pluramycin-type antitumor antibiotic hedamycin has been cloned from Streptomyces griseoruber. Sequence analysis of the 45.6 kb region revealed a variety of unique features such as a fabH homolog (KSIII), an acyltransferase (AT) gene, a set of type I polyketide synthase (PKS) genes, and two putative C-glycosyltransferase genes. As the first report of the cloning of the biosynthetic gene cluster for the pluramycin antibiotics, this work suggests that the biosynthesis of pluramycins utilize an iterative type I PKS system for the generation of a novel starter unit that subsequently primes the type II PKS system. It also implicates the involvement of a second catalytic ketosynthase (KSIII) to regulate this unusual priming step. Gene disruption is used to confirm the importance of both type I and II PKS genes for the biosynthesis of hedamycin.     

Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.     

Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host

Erythromycin A is a potent antibiotic long-recognized as a therapeutic option for bacterial infections. The soil-dwelling bacterium Saccharopolyspora erythraea natively produces erythromycin A from a 55 kb gene cluster composed of three large polyketide synthase genes (each ~10 kb) and 17 additional genes responsible for deoxysugar biosynthesis, macrolide tailoring, and resistance. In this study, the erythromycin A gene cluster was systematically transferred from S. erythraea to E. coli for reconstituted biosynthesis, with titers reaching 10 mg/l. Polyketide biosynthesis was then modified to allow the production of two erythromycin analogs. Success establishes E. coli as a viable option for the heterologous production of erythromycin A and more broadly as a platform for the directed production of erythromycin analogs.     

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

BACKGROUND: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS-PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide-polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. RESULTS: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. CONCLUSION: The blm gene cluster is characterized by a hybrid NRPS-PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide-polyketide metabolites.     

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an aromatic polyketide synthase

BACKGROUND: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. RESULTS: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. CONCLUSIONS: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.     

Micellar electrokinetic chromatographic analysis for in vitro accumulation of anthracyclines enhanced by inhibitors of cell membrane transporter-proteins in cancer cells

Cell membrane transporter-proteins have been partly implicated in lowering the accumulation of drugs in cancer cells, leading to multidrug resistance (MDR). Two cancer cell lines, A549 and RDES, were continuously exposed to subclinical concentration (250 nM) of anthracyclines and micellar electrokinetic chromatography was used to investigate their in vitro accumulation after treatment with inhibitors of membrane transporter-proteins. The four anthracylines [doxorubicin (DOX), epirubicin (EPI), daunorubicin (DNR), and idarubicin (IDA)] were separated within a short analysis time of less than 15 min in borate buffer (80 mM, pH 9.22) containing sodium taurodeoxycholate (35 mM), 2-hydroxypropyl-γ-cyclodextrin (3.5% wt/v), and sodium dodecylsulfate (20 mM). Laser-induced fluorescence was used for detection of the anthracyclines. Three inhibitors, verapamil, cyclosporine A and probenecid, were examined by adding each inhibitor independently or two inhibitors simultaneously to the culture medium. It was found that independent use of each inhibitor leads to more efficient accumulation than combined use of verapamil and probenecid. In addition, the results show that effect of inhibitors on the accumulation of anthracyclines depended on type of cell: in RDES, inhibitors enhanced accumulation of all four anthracyclines, while in A549, inhibitors showed different accumulation behavior for each anthracycline. Generally higher accumulation of anthracyclines was observed in RDES cells than A549, as evidenced by dead cells (7-16%) after 24 h of continuous exposure to subclinical concentration.     

柔红霉素与DNA相互作用的机理研究

本文以循环伏安、光谱电化学和原子力显微镜方法从DNA角度研究柔红霉素与天然鱼精DNA和热变性DNA之间相互作用的机理。并对柔红霉素与鱼精DNA和热变性DNA复合物的组成及复合物的形成常数作了测定。研究发现嵌入作用是柔红霉素和天然DNA之间的主要作用方式；并且柔红霉素和天然DNA之间的作用要强于和热变性单链DNA之间的作用。对这两种复合物的光谱电化学和原子力显微镜研究表明，在体内氧化还原代谢条件下，柔红霉素还原过程中产生的半醌自由基可引发自由基链反应，造成DNA链的解链、断裂等损伤。