RESUMEN
A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.
Asunto(s)
Myxococcales , Sorangium , Sorangium/genética , Sintasas Poliquetidas/genética , Familia de Multigenes , Myxococcales/genéticaRESUMEN
This review details the isolation, biosynthesis, biological activity and synthesis of spiroacetals from the myxobacterium Sorangium cellulosum. The strategies utilised to access the challenging structures and stereochemistry of these natural products are highlighted.
Asunto(s)
Acetales/metabolismo , Productos Biológicos/metabolismo , Compuestos de Espiro/metabolismo , Acetales/química , Productos Biológicos/química , Conformación Molecular , Sorangium/química , Sorangium/metabolismo , Compuestos de Espiro/química , EstereoisomerismoRESUMEN
Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.
Asunto(s)
Glicerol/farmacología , Myxococcales/metabolismo , Myxococcus xanthus/efectos de los fármacos , Myxococcus xanthus/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Espectrometría de Masas , Myxococcus xanthus/metabolismo , Myxococcus xanthus/fisiología , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal/fisiología , Sorangium/química , Sorangium/metabolismo , Esporas Bacterianas , Estrés Fisiológico , Resonancia por Plasmón de Superficie , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea. Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.
Asunto(s)
Comamonadaceae/química , Epotilonas/biosíntesis , Ingeniería Metabólica/métodos , Proteínas Bacterianas/genética , Coenzima A Ligasas/genética , Comamonadaceae/genética , Comamonadaceae/metabolismo , Epotilonas/química , Familia de Multigenes , Plásmidos/genética , Plásmidos/metabolismo , Sintasas Poliquetidas/genética , Policétidos/química , Policétidos/metabolismo , Regiones Promotoras Genéticas , Racemasas y Epimerasas/genética , Sorangium/genéticaRESUMEN
Epothilone is a macrolide secondary metabolite which has the same anticancer effect as paclitaxel. Based on a series of single-factor experiments, four factors, temperature, initial pH, rotation speed, and inoculum quantity, which have the greatest influence on yield, were determined. Four factors were designed and orthogonal experiments were carried out to optimize the fermentation conditions. Finally, the best experimental conditions were obtained as follows: 250 ml flapper triangular flask was used. The yield of epothilone B was 39.76 mg/L at 30â, initial pH = 7.4, rotating speed 200 r/min, inoculation amount 10%, liquid loading amount 50 ml/250 ml, fermentation time 6 days and seed age 60 hr.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/metabolismo , Epotilonas/química , Epotilonas/metabolismo , Antineoplásicos/farmacología , Técnicas de Cultivo de Célula , Epotilonas/farmacología , Fermentación , Concentración de Iones de Hidrógeno , Oxígeno/metabolismo , Metabolismo Secundario , Sorangium/metabolismo , Temperatura , Factores de TiempoRESUMEN
Epothilone, the macrolide compound produced by Sorangium cellulosum, has antitumor activity. Its anti-tumor mechanism is similar to that of paclitaxel, which promotes the polymerization of tubulin and induces apoptosis. Herein, 7-O-ß-d-galactosyl-polyethylene glycol-epothilone B 6 was synthesized. It showed that the toxicity of the synthesized compound was 1/182 of the epothilone B. In addition, compound 6 also had significant anticancer activity under the action of enzyme.