Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.
Biological Products/metabolism* ; Lactams, Macrocyclic/metabolism* ; Multigene Family ; Organisms, Genetically Modified ; Streptomyces/metabolism*

Geldanamycin (Gdm), an inhibitor of heat shock protein 90 (Hsp90), shows antitumor and antivirus bioactivity. Most Geldanamycin biosynthetic genes have been cloned from the genome library of Streptomyces hygroscopicus 17997. In this report, polyketide synthase (pks) gene, mono-oxygenase (gdmM) gene and carbamoyltransferase gene (gdmN) were subjected to inactivation. Three gene disrupted mutants (deltapks, deltagdmM and deltagdmN) were obtained by double crossover. No Geldanamycin production was detected in three mutant strains cultured in fermentation broth. Gene complementation experiments excluded the possible polar effect of gene disruption on other genes. These results confirmed that pks, gdmM and gdmN genes were essential for Geldanamycin biosynthesis.
Benzoquinones ; metabolism ; Carboxyl and Carbamoyl Transferases ; genetics ; Lactams, Macrocyclic ; metabolism ; Mixed Function Oxygenases ; genetics ; Polyketide Synthases ; genetics ; Streptomyces ; genetics ; metabolism

A geldanamycin (GDM) producing strain, Streptomyces hygroscopicus 17997, was isolated from Yunnan China soil by our institute researchers. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (Heat Shock Protein 90) and alter its function. Hsp90 is a chaperone protein involved in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis. So it plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumor and antivirus bioactivity, but the hypato-toxicity and poor solubility in water limits its clinical use. Two GDM derivatives, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), both showing lesser hepato-toxicity, are now in Phase II and Phase I clinic trials. In order to accomplish the structure modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732 bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT gene containing fragments were verified and localized by Southern blot. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. Based on BLAST analysis, this sequence contains 13 possible ORFs and their deduced functions are believed to be involved in GDM production. The ORF1 encoding products show homology (87%) with incomplete sixth module and complete seventh module of PKS, gdmA3, in S. hygroscopicus NRRL 3602. The ORF2-13 gene products are similar to gdmF(9 5%), gdmM(8 8%), gdmN (92%), gdmH (92%), I (93%), J (90%), K (93%), G (96%), gdmO (91%), gdmP (93%) and two transcription regulation genes gdmRI (83%) and gdmRII (90%). The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and to modify the structure of GDM through combinatorial biosynthesis.
Anti-Bacterial Agents ; metabolism ; Benzoquinones ; metabolism ; Carboxyl and Carbamoyl Transferases ; genetics ; Chromosome Mapping ; Cloning, Molecular ; DNA Primers ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; Streptomyces ; genetics ; metabolism

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.
Alcohol Oxidoreductases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; Benzoquinones ; metabolism ; Blotting, Southern ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Genetic Vectors ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; genetics ; physiology ; Polymerase Chain Reaction ; Streptomyces ; genetics ; metabolism

To identify the anti-bacterial compound(s) from Streptomyces hygroscopicus 17997, a geldanamycin producer, silica gel thin layer chromatography (TLC) TLC was used to separate the secondary metabolites of S. hygroscopicus 17997. Compound(s) from the silica gel TLC with anti-Gram positive bacteria activity and becoming red upon color reaction by 2.0 mol/L NaOH was analyzed by HPLC. The UV absorption profile and the retention time of a peak of HPLC were identical to those of authentic elaiophylin. A conserved region of dTDP-glucose-4,6-dehydratase (Tgd) gene was amplified by PCR from the genomic DNA of Streptomyces hygroscopicus 17997. DNA sequence analysis of the amplified DNA fragment indicated that it should be the tgd gene of elaiophylin biosynthetic gene cluster. These results implied that the compound in the peak of HPLC was elaiophylin, a macrodiolide antibiotic. The compound was then confirmed to be elaiophylin by LC-(+)-ESI-MS, which revealed that Streptomyces hygroscopicus 17997 was an elaiophylin producer. At the same time, a fast procedure, which consisted of silica gel TLC, color reaction, HPLC, PCR detection and DNA sequence analysis of tgd gene, and LC-(+)-ESI-MS, was established for rapid identification of elaiophylin and its producer.
Benzoquinones ; metabolism ; Chromatography, Liquid ; methods ; DNA, Bacterial ; genetics ; Hydro-Lyases ; genetics ; Lactams, Macrocyclic ; metabolism ; Macrolides ; analysis ; isolation & purification ; metabolism ; Mass Spectrometry ; methods ; Sequence Analysis, DNA ; Streptomyces ; genetics ; isolation & purification ; metabolism

Two LAL family regulatory genes, gdmRI and gdmRII, were identified in the geldanamycin biosynthetic gene cluster of Streptomyces hygroscopicus 17997. Disruption of the two regulatory genes resulted in absolute elimination of geldanamycin biosynthesis. The complementation experiments using a single wild-type gene could restore geldanamycin production. These results indicated that both gdmRI and gdmRII were positive regulatory genes of the geldanamycin biosynthesis.
Anti-Bacterial Agents ; biosynthesis ; Benzoquinones ; metabolism ; Gene Expression Regulation, Bacterial ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Lactams, Macrocyclic ; metabolism ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Repressor Proteins ; genetics ; Streptomyces ; genetics ; metabolism ; Trans-Activators ; genetics

OBJECTIVETo investigate the antitumor effect of a benzoquinone ansamycin antibiotic, geldanamycin (GA), against HER2 /neu tyrosine kinase-overexpressing human breast cancer cell line SKBr3.

METHODSTo evaluate the antitumor activity of GA, the degradation of HER2 /neu tyrosine kinase in GA-treated SKBr3 cells was analyzed by Western blotting, their proliferation assessed using MTT assay, and the cell cycle distribution identified by flow cytometry. RT-PCR and Real-time PCR were employed to detect cyclin D1 mRNA expression and cell culture inserts model was used to evaluate the motility of the cells.

RESULTSGA induced a dose- and time-dependent degradation of HER2 /neu tyrosine kinase and cell proliferation inhibition. GA treatment obviously decreased the survival rates of the cancer cells, leading also to a dose-dependent G(1) arrest. The antitumor effects of GA proved to be relevant with declined transcription of cyclin D1. The GA-treated cells also exhibited reduced motility.

CONCLUSIONGA can efficiently destabilize HER2 /neu tyrosine kinase and inhibit the proliferation and motility of human breast cancer cell line SKBr3 overexpressing HER2 /neu tyrosine kinase.

Anti-Bacterial Agents ; pharmacology ; Benzoquinones ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; physiopathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Female ; Gene Expression ; drug effects ; Humans ; Lactams, Macrocyclic ; pharmacology ; Receptor, ErbB-2 ; genetics ; metabolism

OBJECTIVE: To investigate the role of focal adhesion kinase (FAK) in extracellular signal-regulated kinase (ERK) signaling pathway mediated invadsion of trophoblasts. METHODS: We established a human extravillous cytotrophoblasts in vitro invasion model. Different concentrations of herbimycin A(FAK inhibitor)and PD98059 (ERK inhibitor) were given to observe the influence on the growth of trophoblast cells, FAK, ERK phosphorylation, and trophoblast invasion abilities. RESULTS: The expression of phosphorylated FAK in the extravillous cytotrophoblasts (EVCT) was inhibited by herbimycin A in a concentration-dependent manner and expression of phosphorylated ERK1/2 was also partially reduced. PD98059 had no effect on the expression of phosphorylated FAK. Herbimycin A and PD98059 suppressed the in vitro invasion of EVCT to various degrees. CONCLUSION ERK signaling pathway may be the common pathway for many invasive signals,and play a key role in the regulation of trophoblast invasion.
Benzoquinones ; pharmacology ; Cell Division ; physiology ; Cell Movement ; physiology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Phosphorylation ; Rifabutin ; analogs & derivatives ; Signal Transduction ; physiology ; Trophoblasts ; cytology ; physiology

OBJECTIVETo study the effect of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on cell proliferation and apoptosis of human cancer SGC-7901 cells and explore the mechanisms.

METHODSThe inhibitory effect of 17-AAG on the proliferation and morphology of SGC-7901 cells was assessed with MTT assay and DNA-PI staining, respectively. Flow cytometry was employed to analyze the changes in cell cycle and apoptosis of the cells following 17-AAG exposure. The cellular expression of Fas protein was detected by immunohistochemistry.

RESULTS17-AAG significantly suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner. After treatment with 17-AAG for 48 h, SGC-7901 cells showed cell cycle arrested at G(2)/M stage, and the cell apoptosis rate increased with the 17-AAG concentration. The expression of Fas protein in the cytoplasm of SGC-7901 cells increased gradually with the increase of 17-AAG concentration.

CONCLUSION17-AAG can induce apoptosis, alters the cell cycle distribution and up-regulates the expression of Fas protein in SGC-7901 cells to suppress the cell proliferation.

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Stomach Neoplasms ; pathology ; fas Receptor ; metabolism

OBJECTIVETo explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells.

METHODSKasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR.

RESULTS17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased.

CONCLUSION17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.

Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics