球孢链霉菌一种抗生素生物合成基因的核苷酸序列分析

C-1027是一种具有极强抗肿瘤活性的新型抗生素,由球孢链霉菌（Streptomycesglobisporus）产生。F2DNA片段含有一种编码C－1027生物合成的基因。以质粒pUC18为载体,对其进行了亚克隆,并对该片段进行了核苷酸序列分析,发现一编码122个氨基酸的开放阅读框架,经检索可能是一种新的序列。     

球孢链霉菌质粒pSGL1的性质研究

链霉菌质粒pSGL1是本实验室在球孢链霉菌（Streptomycelglobisporus）中发现的新的质粒,它可以在变铅青链霉菌中复制。用缺失分析确定了pSGL1的基本复制区位于一个2．0kb的片段上。pSGL1能够与pIJ101相容。pSGL1是一个高拷贝质粒,拷贝数约为70～250。在对质粒进行分析的过程中得到的一些衍生质粒如pSGLN和pSGLS3等可以作为新的链霉菌基因克隆载体,并可用于构建新的高效分泌表达的链霉菌载体。     

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.     

Cloning and nucleotide sequencing of the antitumor antibiotic C-1027 apoprotein gene.

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met1---Ala33 leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mcmA) while the flanking regions had little homology with the corresponding ones of mcmA, except some homology at the -10th and -35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

Characterization of SgcE6, the flavin reductase component supporting FAD-dependent halogenation and hydroxylation in the biosynthesis of the enediyne antitumor antibiotic C-1027

The C-1027 enediyne antitumor antibiotic from Streptomyces globisporus possesses an ( S )-3-chloro-5-hydroxy-β-tyrosine moiety, the chloro- and hydroxy-substituents of which are installed by a flavin-dependent halogenase SgcC3 and monooxygenase SgcC, respectively. Interestingly, a single flavin reductase, SgcE6, can provide reduced flavin to both enzymes. Bioinformatics analysis reveals that, similar to other flavin reductases involved in natural product biosynthesis, SgcE6 belongs to the HpaC-like subfamily of the Class I flavin reductases. The present study describes the steady-state kinetic characterization of SgcE6 as a strictly NADH- and FAD-specific enzyme.     

Cloning,expression and characterization of gene <Emphasis Type="Italic">sgcD</Emphasis> involved in the biosynthesis of novel antitumor lidamycin

Lidamycin with high antitumor activity is a novel enediyne antitumor antibiotic producedby Streptomyces globisporus C1027. The 75 kb biosynthesis gene cluster of lidamycin containing 33 open reading frames has been cloned from S. globisporus C1027. In this paper,the function ofsgcD (ORF24) is investigated. Gene disruption experiment proved that sgcD is involved in lidamy-cin biosynthesis. With homologous comparing analysis, we deduce that sgcD codes aminomutasecatalyzing α-tyrosine to β-tyrosine which is one motif for lidamycin. To identify the function of en-zyme coded by sgcD, sgcD is cloned into vector pET30a for inducing expression and the activity ofexpression product is analyzed. The result showed that the expression product ofsgcD has theactivity of aminomutase. Aminomutase coded by sgcD is the first characterized enzyme involved inthe biosynthesis of enediyne antitumor antibiotics. Our research will be helpfulto clarifying thebiosynthesis mechanism of such kind of antibiotic and to producing new antitumorcompounds.     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin, an antitumor antibiotic composed of a macro-molecule peptide and enediyne chromophore[1] and originally named C1027, is produced by Streptomyces globisporus C1027 isolated from the soil in Qianjiang County, Hubei Province, China. It has extremely high antitu- mor activity, which has been proved to be the highest among antitumor compounds[2], being 1000- fold higher than that of adriamycin commonly used in clinic. The structure of lidamycin consists of an acid apoprotein and a chr…     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases

The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027. Moreover, phosphorylation of p53 Ser-15 induced by C-1027 was independent of ATM, ATR, or DNA-PK function. We have concluded that the model based on IR studies cannot always be directly applied to DNA damage induced by other strand-scission agents.     

DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase.

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.     

Design of Streptomyces nogalater LV65 strains with higher synthesis of nogalamicin using regulatory genes

Influence of cloned regulatory genes on biosynthesis of nogalamicin by Streptomyces nogalater LV65 strains has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into the cells of wild type strain of S. nogalater LV65 resulted in higher synthesis of nogalamicin. A similar effect was observed at heterologous expression of gene ppGpp of synthetase relA cloned in S. coelicolor A3(2). Heterologous expression of genes absA2from S. ghanaensis ATCC14672 and lndyR from genome S. globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

Interspecies conjugation of Escherichia coli-Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives]

Streptomyces globisporus 1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 from Escherichia coli into S. globisporus 1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporus chromosome and is stably inherited under nonselective conditions. pSET152+ exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50-90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in ex-conjugants was 120-150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporus was developed. The effectivity of this system was shown in the example of disruption of the lndA gene encoding the key enzyme of LE synthesis (beta-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

Background  

C-1027, produced by Streptomyces globisporus C-1027, is one of the most potent antitumoral agents. The biosynthetic gene cluster of C-1027, previously cloned and sequenced, contains at least three putative regulatory genes, i.e. sgcR1, sgcR2 and sgcR3. The predicted gene products of these genes share sequence similarities to StrR, regulators of AraC/XylS family and TylR. The purpose of this study was to investigate the role of sgcR3 in C-1027 biosynthesis.     

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.     

Generation of landomycin D-producing strainStreptomyces globisporus LD3

Rhodinosyl transferase gene lndGT4, governing the conversion of the disaccharide oligoketide ('polyketide') landomycin D into a trisaccharide derivative landomycin E, was deleted in Streptomyces globisporus 1912 genome. Possible resistance mechanisms that protect the resulting landomycin D-producing mutant strain S. globisporus LD3 against the toxic action of landomycins were determined.     

Cloning and Nucleotide Sequencing of the Antitumor Antibiotic C-1027 Apoprotein Gene

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met¹—Ala³³ leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, ¹⁾ (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mernA) while the flanking regions had little homology with the corresponding ones of mernA, except some homology at the – 10th and – 35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.     

The use of PCR for detecting genes that encode type I polyketide synthases in genomes of actinomycetes

PCR screening of type I polyketidesynthase genes (PKS) was conducted in genomes of actinomycetes, producers of antibiotics. Some DNA fragments from the Streptomyces globisporus 1912 strain, a producer of a novel angucycline antibiotic landomycin E, were amplified. These fragments shared appreciable homology with type I PKS controlling the biosynthesis of polyene antibiotics (pymaricin and nistatin). The cloned regions were used to inactivate putative type I PKS genes in S. globisporus 1912. Strains with inactivated genes of PKS module do not differ from the original strain in the spectrum of synthesized polyketides. Apparently, these are silent genes, which require specific induction for their expression. The method of PCR screening can be used in a large-scale search for producers of new antibiotics.