Product Geldanamycin Research Articles

Discovery of Bioactive Metabolites by Acidic Stress to a Geldanamycin Producer, Streptomyces samsunensis.

In an effort to activate silent biosynthetic gene clusters, Streptomyces samsunensis DSM42010, a producer of geldanamycin, was cultured at four different pHs (4.5, 5.4, 6.6, and 7.4). An acidic culture condition (pH 5.4) was selected for a chemical investigation since S. samsunensis showed a different metabolic profile compared to when it was cultured under other conditions. Seven new (1-7) and four known (8-11) compounds were isolated from these cultures. The structures of the isolated compounds were determined by spectroscopic techniques and chemical derivatization. Relative and absolute configurations of the new compounds (1-5) were established using JBCA, PGME method, advanced Marfey's method, modified Mosher's method, and comparison of observed and calculated ECD data. Interestingly, compounds 1-3 were truncated versions of geldanamycin, and compound 4 was also deduced to originate from geldanamycin. Compound 5 was composed of 3-methyltyrosine and 6-hydroxy-2,4-hexadienoic acid connected through an amide bond. Compounds 6 and 7 were dihydrogenated forms of geldanamycin with a hydroxy substitution. It is possible that culturing this strain under acidic conditions interfered to some degree with the geldanamycin polyketide synthase, leading to production of truncated versions as well as analogues of geldanamycin. Compounds 1, 8, and 9 showed significant antivirulence activity, inhibiting production of α-toxin by methicillin-resistant Staphylococcus aureus without growth attenuation and global regulatory inhibition; compounds 1, 8, and 9 may become promising α-toxin-specific antivirulence leads with less risk of resistance development.

Regulation of Geldanamycin Biosynthesis by Cluster-Situated Transcription Factors and the Master Regulator PhoP.

Geldanamycin and the closely related herbimycins A, B, and C are benzoquinone-type ansamycins with antitumoral activity. They are produced by Streptomyces hygroscopicus var. geldanus, Streptomyces lydicus and Streptomyces autolyticus among other Streptomyces strains. Geldanamycins interact with the Hsp-90 chaperone, a protein that has a key role in tumorigenesis of human cells. Geldanamycin is a polyketide antibiotic and the polyketide synthase contain seven modules organized in three geldanamycin synthases genes named gdmAI, gdmAII, and gdmAIII. The loading domain of GdmI activates AHBA, and also related hydroxybenzoic acid derivatives, forming geldanamycin analogues. Three regulatory genes, gdmRI, gdmRII, and gdmRIII were found associated with the geldanamycin gene cluster in S. hygroscopicus strains. GdmRI and GdmRII are LAL-type (large ATP binding regulators of the LuxR family) transcriptional regulators, while GdmRIII belongs to the TetR-family. All three are positive regulators of geldanamycin biosynthesis and are strictly required for expression of the geldanamycin polyketide synthases. In S. autolyticus the gdmRIII regulates geldanamycin biosynthesis and also expression of genes in the elaiophylin gene cluster, an unrelated macrodiolide antibiotic. The biosynthesis of geldanamycin is very sensitive to the inorganic phosphate concentration in the medium. This regulation is exerted through the two components system PhoR-PhoP. The phoRP genes of S. hygroscopicus are linked to phoU encoding a transcriptional modulator. The phoP gene was deleted in S. hygroscopicus var geldanus and the mutant was unable to grow in SPG medium unless supplemented with 5 mM phosphate. Also, the S. hygroscopicus pstS gene involved in the high affinity phosphate transport was cloned, and PhoP binding sequences (PHO boxes), were found upstream of phoU, phoRP, and pstS; the phoRP-phoU sequences were confirmed by EMSA and nuclease footprinting protection assays. The PhoP binding sequence consists of 11 nucleotide direct repeat units that are similar to those found in S. coelicolor Streptomyces avermitilis and other Streptomyces species. The available genetic information provides interesting tools for modification of the biosynthetic and regulatory mechanisms in order to increase geldanamycin production and to obtain new geldanamycin analogues with better antitumor properties.

Improved PKS Gene Expression With Strong Endogenous Promoter Resulted in Geldanamycin Yield Increase.

The type I polyketide geldanamycin is a potent anti-tumor reagent. Its biosynthesis includes three steps: the biosynthesis of precursors, such as 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase (PKS) chain extension, and the post-PKS modifications. According to the genomic and transcriptomic analysis, the PKS chain extension was deduced to be the rate-limiting step for geldanamycin production in Streptomyces hygroscopicus XM201. In order to improve the expression of PKS genes, a strong endogenous promoter 5063p was obtained based on the transcriptomic analysis and XylE enzymatic assay. By replacing the native PKS promoter gdmA1p with 5063p, the expression of the PKS genes during geldanamycin fermentation was increased by 4-141-folds, and the geldanamycin yield was increased by 39%. Interestingly, AHBA feeding experiment showed that the supply of AHBA in turn become a new rate-limiting factor for geldanamycin production. Further combined overexpression of the 6-gene AHBA biosynthetic cassette and PKS genes increased the yield of geldanamycin by 88%, from 773 mg L-1 of the wild-type to 1450 mg L-1 in the derived strain. Our results suggested that improved expression of all PKS genes in a particular biosynthetic gene cluster is important for the yield increase of the corresponding polyketide natural product.

Streptomyces cameroonensis sp. nov., a Geldanamycin Producer That Promotes Theobroma cacao Growth.

The taxonomy of an actinobacterial strain, designated JJY4T, was established using a polyphasic approach. JJY4T was isolated from the rhizosphere of Chromolaena odorata in Yaoundé (Cameroon) during a project for the selection of biological control agents. Strain JJY4T exhibited antimicrobial activities against bacteria, fungi, and oomycetes. Strain JJY4T also exhibited the traits of plant growth-promoting rhizobacteria such as the solubilization of inorganic phosphate, production of siderophores and indole-3-acetic acid, and 1-aminocyclopropane-1-carboxylate deaminase activity. In planta assays performed on cocoa plantlets confirmed that strain JJY4T exhibited strong abilities to promote plant growth and protect against Phytophthora megakarya, the main causal agent of cocoa pod rot. The formation of rugose-ornamented spores in spiral spore chains by strain JJY4T is a typical feature of members found in the Streptomyces violaceusniger clade and, similar to some members of the clade, strain JJY4T produces geldanamycin. A phylogenetic analysis based on 16S rRNA gene sequences confirmed this classification and suggests that strain JJY4T be added to the subclade constituted of the type strains Streptomyces malaysiensis DSM 41697T and Streptomyces samsunensis DSM 42010T. However, DNA–DNA relatedness and physiological characteristics allowed for the differentiation of strain JJY4T from its closest phylogenetic relatives. Based on these results, strain JJY4T (=NRRL B-65369, =NBRC 112705) appears to represent a novel species in the S. violaceusniger clade for which the proposed name is Streptomyces cameroonensis sp. nov.

Synthesis of a cytotoxic ansamycin hybrid.

The synthesis of a new ansamycin macrolactam derivative that contains an ansa chain based on ansamitocin and an aromatic core related to geldanamycin is reported. The selective introduction of the cyclic carbamoyl group at C7 and C9 relies on a biotransformation using a mutant strain of S. hygroscopicus, the geldanamycin producer. The ansamycin hybrid forms atropisomers that differ in their antiproliferative activity toward cancer cells.

Bioreduction of Aryl Azides during Mutasynthesis of New Ansamitocins

Supplementing a culture of a mutant strain of Actinosynnema pretiosum that is unable to biosynthesize aminohydroxy benzoic acid (AHBA), with 3-azido-5-hydroxy-benzoic acid and 3-azido-5-amino-benzoic acid, unexpectedly yielded anilino ansamitocins instead of the expected azido derivatives. This is the first example of the bioreduction of organic azides. The unique nature of these results was demonstrated when 3-azido-5-amino-benzoic acid was fed to the corresponding AHBA blocked mutant of Streptomyces hygroscopicus, the geldanamycin producer. This mutasynthetic experiment yielded the fully processed azido derivative of geldanamycin.

Heterologous Expression of a Putative K+/H+ Antiporter of S. coelicolor A3(2) Enhances K+, Acidic-pH Shock Tolerances, and Geldanamycin Secretion

Heterologous expression of a putative K+/H+ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to K+ stress, acidic-pH shock, and/or geldanamycin production under K+ stress. In a series of K+ extrusion experiments with sha4-carrying E. coli deficient in the K+/H+ antiporter, a restoration of impaired K+ extrusion activity was observed. Based on this, it was concluded that sha4 was a true K+/H+ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to K+ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in K+ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under K+ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

Broad Substrate Specificity of the Amide Synthase in S. hygroscopicus—New 20-Membered Macrolactones Derived from Geldanamycin

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.

A new method for rapid identification of ansamycin compounds by inactivating KLM gene clusters in potential ansamycin-producing actinomyces

In this study, we explored the possibility of construction of a 'universal targeting vector' by Red/ET recombination to inactivate L gene encoding 3-amino-5-hydroxybenzoic acid (AHBA)-oxidoreductase in AHBA biosynthetic gene cluster to facilitate the detection of ansamycins production in actinomycetes. Based on the conserved regions of linked AHBA synthase (K), oxidoreductase (L) and phosphatase (M) gene clusters, degenerate primers were designed and PCR was performed to detect KLM gene clusters within 33 AHBA synthase gene-positive actinomycetes strains. Among them, 22 KLM gene cluster-positive strains were identified. A 'universal targeting vector' was further constructed using the 50-nt homologous sequences chosen from four strains internal L gene in KLM gene clusters through Red/ET recombination. The L gene from nine of the KLM gene cluster-positive actinomycetes strains was inactivated by insertion of a kanamycin (Km) resistance marker into its internal region from the 'universal targeting vector'. By comparison of the metabolites produced in parent strains with those in L gene-inactivated mutants, we demonstrated the possible ansamycins production produced by these strains. One strain (4089) was proved to be a geldanamycin producer. Three strains (3-20, 7-32 and 8-32) were identified as potential triene-ansamycins producers. Another strain (3-27) was possible to be a streptovaricin C producer. Strains 24-100 and 4-124 might be served as ansamitocin-like producers. The results confirmed the feasibility that a 'universal targeting vector' could be constructed through Red/ET recombination using the conserved regions of KLM gene clusters to detect ansamycins production in actinomycetes. The 'universal targeting vector' provides a rapid approach in certain degree to detect the potential ansamycin producers from the 22 KLM gene cluster-positive actinomycetes strains.

Genome shuffling enhances biocontrol abilities of Streptomyces strains against two potato pathogens

To employ the genome shuffling technique for improving the phenotype of a biocontrol control agent of the genus Streptomyces. Two rounds of genome shuffling (GS) were carried out with Streptomyces melanosporofaciens EF-76, a geldanamycin producer. Six fusants that showed optimized in vitro antagonistic activity against Streptomyces scabies or Phytophthora infestans, two important pathogens of potato crops, were selected. All selected fusants retained the capacity to produce geldanamycin, but none overproduced this antibiotic. The higher antagonism ability appeared to result from a diversification of secreted metabolites. Seven or eight metabolites were detected in the HPLC profiles of parental strains, whereas 12-15 were detected in fusant strains. Biocontrol assays revealed that four of six fusants protected tubers more efficiently than parental strains. GS emerged as an elegant and rapid tool to optimize the antagonistic ability of Streptomyces strains. Optimization of the in vitro antagonistic activity against plant pathogens appears to be an effective approach to select for improved biocontrol agents. The enhanced phenotype did not depend on an overproduction of a specific antibiotic but rather on the secretion of a wider variety of secondary metabolites. Improved capacities of a biocontrol agent compensate for the lack of efficient chemical control of potato scab.

Effect of biopolymers on geldanamycin production and biocontrol ability of Streptomyces melanosporofaciens strain EF-76

The biocontrol agent Streptomyces melanosporofaciens strain EF-76 produces geldanamycin, an ansamycin antibiotic which displays antagonistic activity towards several Gram-positive bacteria and fungi. Biopolymers [chitin, carboxymethylcellulose (CMC) and xylan] or N-acetylglucosamine were added to S. melanosporofaciens culture media and the effect of these additional carbon sources on geldanamycin production was analysed. Geldanamycin biosynthesis was only stimulated in the presence of 1 g L−1 of chitin. Higher concentrations of chitin, as well as the presence of CMC or xylan, did not improve antibiotic production. N-acetylglucosamine, the final degradation product of chitin, did not stimulate geldanamycin production at all concentrations tested. Nevertheless, the addition of chitin in culture media had no or a negative impact on the antagonistic activity of EF-76 towards the pathogen Streptomyces scabies. Furthermore, the effect of chitin, CMC and xylan on the biocontrol ability of EF-76 against S. scabies was tested on radish seedlings. None of the polymers affected the biocontrol efficiency of EF-76 under gnotobiotic conditions. However, the addition of CMC or xylan to plant growth medium protected radish seedlings against S. scabies infection.

Identification of Three Positive Regulators in the Geldanamycin PKS Gene Cluster of Streptomyces hygroscopicus JCM4427

In the Streptomyces hygroscopicus JCM4427 geldanamycin biosynthetic gene cluster, Five putative regulatory genes were identified by protein homology searching. Among three of those genes, gel14, gel17, and gel19, are located downstream of polyketide synthase genes. Gel14 and Gel17 are members of the LAL family of transcriptional regulators, including an ATP/GTP-binding domain at the N-terminus and a DNA binding helix-turn-helix domain at the C-terminus. Gel19 is a member of the TetR family transcriptional regulators, which generally act to repress transcription. To verify the biological significance of the putative regulators in geldanamycin production, they were individually characterized by gene disruption, genetic complementation and transcriptional analyses. All three genes were confirmed as positive regulators of geldanamycin production. Specifically, Gel17 and Gel19 are required for gel14 as well as gelA gene expression.

The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus

The diverse morphology of the filamentous organism Streptomyces hygroscopicus var. geldanus was characterised by image analysis under various environmental conditions. In the presence of surfactant compounds, a significant decrease in the mean pellet diameter was observed. Cell aggregation was also influenced by spore inoculum level, with high concentrations reducing pellet size. In addition, the dispersion of pellets was found to increase with the inclusion of glass beads to submerged shake-flask cultures. In all cases, production of the secondary metabolite geldanamycin was determined to be dependent on the morphological profile of the organism, with a concomitant increase of 88% in geldanamycin yield observed as the mean pellet diameter was reduced by 70%. Thus, to maximise the yield of geldanamycin, it is necessary to limit pellet formation in Streptomyces hygroscopicus var. geldanus to an appropriate size.

A New Post-PKS Modification Process in the Carbamoyltransferase Gene Inactivation Strain of Streptomyces hygroscopicus 17997

Genetic manipulation of geldanamycin (GDM) producer Streptomyces species is a rational approach to understand biosynthesis processes and create new analogues. In this study, the carbamoyltransferase gene gdmN was inactivated by insertion of an apramycin-resistance gene aac3 (IV) into the genome of the geldanamycin-producing strain Streptomyces hygroscopicus 17997. GDM analogues produced by this mutant strain were isolated and characterized, such as new compound 4,5-dihydro-7-O-descarbamoyl-7-hydroxy-19-O-glycylgeldanamycin. This compound could be converted to compound 4,5-dihydro-19-O-glycylgeldanamycin, another new GDM analogue, by a strain of Streptomyces hygroscopicus 17997 in which the GDM-pks was inactivated. These new compounds exhibited reductions of cytotoxicity against HepG2 cancer cells, but increases of aqueous solubility. These results suggest that a new post-polyketide synthase modification was involved in this process to produce new GDM analogues.

Regulatory Genes of Geldanamycin Biosynthesis

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.

The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997

The recent sequencing of the DNA region of the geldanamycin post-polyketide synthase (PKS) modification gene clusters revealed the presence of two regulatory genes: gdmRI (2,907 bp) and gdmRII (2,766 bp). The deduced products of gdmRI and gdmRII (968 and 921 amino acid residues, respectively) were identified as homologues of the LuxR transcriptional regulatory proteins. Inactivation by gene replacement of gdmRI or gdmRII in the Streptomyces hygroscopicus 17997 genome resulted in a complete loss of geldanamycin production. Complementation by a plasmid carrying gdmRI or gdmRII restored geldanamycin production, suggesting that the products of these two regulatory genes are positive regulators that are required for geldanamycin biosynthesis. The gdmRI transcript was detected in the DeltagdmRII mutant, and the gdmRII was detected in the DeltagdmRI mutant, indicating that the two genes are transcribed independently and do not regulate each other. Time course of gene expression analysis by RT-PCR of the geldanamycin biosynthetic genes showed that the transcription of gdmRI and gdmRII correlates with that of genes involved in polyketide biosynthesis, but not with the post-PKS modification gene gdmN, whose transcription is initiated earlier. gdmRI or gdmRII gene disruptants did not transcribe the polyketide biosynthetic related genes pks, gdmF, and gdnA-O-P, but did trancribe gdmN. These results demonstrated that gdmRI and gdmRII are pathway-specific positive regulators that control the polyketide biosynthetic genes in geldanamycin biosynthesis, but not the post-PKS modification gene, gdmN.

Construction and Cultivation of Genetically-engineered Strain to Improve Geldanamycin Production

To enhance Streptomyces hygroscopicus 17997, a geldanamycin(Gdm)-producing strain, in fermentation yield, the naphthalenic 3-amino-5-hydroxybenzoic acid (AHBA) biosynthetic genes ( shnSOP), encoding the products that share the common biosynthetic substrates with Gdm, were disrupted by homologous recombination technology. The resulting mutant strain (Δ SOP) was cultivated on a solid medium, and the number of spores collected from the plates was calculated from 5 to 14 days, and the yield of Gdm was measured by high-performance liquid chromatography. The Gdm fermentation yield of Δ SOP strain was 185% more than that of the parent strain. On solid medium, the spore growth of Δ SOP strain experienced two cycles; the second generation of spores produced a higher yield of Gdm.

Cloning and analysis of geldanamycin partial biosynthetic gene cluster of streptomyces hygroscopicus 17997

A geldanamycin (GDM)-producing strain, Streptomyces hygroscopicus 17997, was isolated from the soil of Yunnan, China, by the researchers of the Institute of Medicinal Biotechnology, CAMS & PUMC. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (heat shock protein 90) and alter its function. Hsp90 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 antitumoral and antiviral bioactivity, but the hypatotoxicity and poor solubility in water limit its clinical use. To accomplish the structural 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 the 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-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356 kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. According to BLAST analysis, this sequence contains 13 possible ORFs, and they are believed to be involved in GDM production. The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and the modification of the structure of GDM through combinatorial biosynthesis.

Production of Ansamycin Polyketide Precursors in Escherichia coli

For the heterologous production of ansamycin polyketides such as rifamycin and geldanamycin in Escherichia coli, a number of unusual but important tools must be engineered into the bacterium. Here we demonstrate efficient production of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and the methoxymalonyl extender unit in E. coli. Previous work has demonstrated the production of the ansamycin starter unit AHBA in E. coli in low yield. It was shown that the low yield is primarily due to acetylation of AHBA into N-acetyl-AHBA. Three methods for minimizing this side reaction were evaluated. First, a putative N-arylamine-acetyltransferase (NAT) was deleted from the E. coli chromosome, although this did not alter N-acetyl-AHBA production. Next, E. coli grown in media devoid of glucose yielded a nearly equal mixture of AHBA and N-acetyl-AHBA. Lastly, the NAT inhibitor glycyrrhizic acid was shown to further inhibit the acetylation reaction. The entire set of genes for synthesizing the methoxymalonyl extender unit was transferred from the geldanamycin producer Streptomyces hygroscopicus into E. coli. The pathway specific ACP isolated from the resulting recombinant strain was found to predominantly occur as methyoxymalonyl-ACP. Together, these findings set the stage for engineered biosynthesis of ansamycin polyketides in E. coli.

Identification of Streptomyces and Kitasatospora strains from Thai soils with geldanamycin production strain

Five strains of actinomycetes were isolated from soils in Thailand. PNK1–3, PNK1–5, TT2–9 and KN-6 were belonged to Streptomyces and FLM-2 was Kitasatospora based on their phenotypic and chemotaxonomic characteristics. Phylogenetic analysis using 16S rDNA sequences revealed that PNK1–3 and PNK1–5 were identified as S. hygroscopicus (99.7%), TT2–9 was closed to S. nogalater DSM 40546T (98.0%), KN-6 was S. purpurascens (99.8%); and FLM-2 showed highest similarity with K. kifunensis LMG 19957T (97.6%). The selected strain, PNK1–3 produced geldanamycin that showed inhibition on the growth of Gram-positive bacteria and strong cytotoxicity against KB and BC cells lines. Its taxonomic characterization has been described.