Actinosynnema pretiosum
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PROVIDER: PRJNA792421 | ENA |
REPOSITORIES: ENA
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Project description:Actinosynnema is a small but well-known genus of actinomycetes for production of ansamitocin, the payload component of antibody-drug conjugates against cancers. However, the secondary metabolite production profile of Actinosynnema pretiosum ATCC 31565, the most famous producer of ansamitocin, has never been fully explored. Our antiSMASH analysis of the genomic DNA of Actinosynnema pretiosum ATCC 31565 revealed a NRPS-PKS gene cluster for polyene macrolactam. The gene cluster is very similar to gene clusters for mirilactam and salinilactam, two 26-membered polyene macrolactams from Actinosynnema mirum and Salinispora tropica, respectively. Guided by this bioinformatics prediction, we characterized a novel 26-membered polyene macrolactam from Actinosynnema pretiosum ATCC 31565 and designated it pretilactam. The structure of pretilactam was elucidated by a comprehensive analysis of HRMS, 1D and 2D-NMR, with absolute configuration of chiral carbons predicted bioinformatically. Pretilactam features a dihydroxy tetrahydropyran moiety, and has a hexaene unit and a diene unit as its polyene system. A preliminary antibacterial assay indicated that pretilactam is inactive against Bacillus subtilis and Candida albicans.
| S-EPMC6631532 | BioStudies
Project description:Actinosynnema pretiosum ATCC 31280 is the producer of antitumor agent ansamitocin P-3 (AP-3). Understanding of the AP-3 biosynthetic pathway and the whole metabolic network in A. pretiosum is important for the improvement of AP-3 titer. In this study, we reconstructed the first complete Genome-Scale Metabolic Model (GSMM) Aspm1282 for A. pretiosum ATCC 31280 based on the newly sequenced genome, with 87% reactions having definite functional annotation. The model has been validated by effectively predicting growth and the key genes for AP-3 biosynthesis. Then we built condition-specific models for an AP-3 high-yield mutant NXJ-24 by integrating Aspm1282 model with time-course transcriptome data. The changes of flux distribution reflect the metabolic shift from growth-related pathway to secondary metabolism pathway since the second day of cultivation. The AP-3 and methionine metabolisms were both enriched in active flux for the last two days, which uncovered the relationships among cell growth, activation of methionine metabolism, and the biosynthesis of AP-3. Furthermore, we identified four combinatorial gene modifications for overproducing AP-3 by in silico strain design, which improved the theoretical flux of AP-3 biosynthesis from 0.201 to 0.372 mmol/gDW/h. Upregulation of methionine metabolic pathway is a potential strategy to improve the production of AP-3.
| S-EPMC6070911 | BioStudies
Project description:Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual "methoxymalonate" polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.
| S-EPMC123004 | BioStudies
Project description:Actinosynnema pretiosum subsp. pretiosum Genome sequencing and assembly
| PRJNA471600 | ENA
Project description:In submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for secondary metabolites production. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. In order to identify genes involved in the early mycelial fragmentation, the total RNAs of mycelia collected at 15, 18, and 24 h were extracted and subjected to transcriptome sequencing using RNA-seq technology.Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. Overall design: Exploration of the expression profile of genes in different mycelial morphology and search for mycelial fragmentation related genes. Three samples are analyzed.
2020-05-25 | GSE151010 | GEO
Project description:Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards ?-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of ?-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM_5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.
| S-EPMC7277737 | BioStudies
Project description:Actinosynnema pretiosum DSM 44132
| PRJNA262061 | ENA
Project description:Actinosynnema pretiosum subsp. pretiosum strain ATCC 31280 ansamitocin gene cluster I
| PRJNA361200 | ENA
Project description:Actinosynnema pretiosum subsp. pretiosum strain ATCC 31280 ansamitocin gene cluster II
| PRJNA362988 | ENA