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Antimicrobial resistance is expected to increase mortality rates by up to several million deaths per year by 2050 without new treatment options at hand. Recently, we characterized the pharmacokinetic (PK) and pharmacodynamic properties of two atypical tetracyclines, chelocardin (CHD) and amidochelocardin (CDCHD) that exhibit no cross-resistance with clinically used antibacterials. Both compounds were preferentially renally cleared and demonstrated pronounced effects in an ascending urinary tract infection model against E. coli. Renal drug transporters are known to influence clearance into the urine. In particular, inhibition of apical transporters in renal tubular epithelial cells can lead to intracellular accumulation and potential cell toxicity, whereas inhibition of basolateral transporters can cause a higher systemic exposure. Here, selected murine and human organic cation (Oct), organic anion (Oat), and efflux transporters were studied to elucidate interactions with CHD and CDCHD underlying their PK behavior. CHD exhibited stronger inhibitory effects on mOat1 and mOat3 and their human homologues hOAT1 and hOAT3 compared to CDCHD. While CHD was a substrate of mOat3 and mOct1, CDCHD was not. By contrast, no inhibitory effect was observed on Octs. CDCHD rather appeared to foster enhanced substrate transport on mOct1. CHD and CDCHD inhibited the efflux transporter hMRP2 on the apical side. In summary, the substrate nature of CHD in conjunction with its autoinhibition toward mOat3 rationalizes the distinct urine concentration profile compared to CDCHD that was previously observed in vivo. Further studies are needed to investigate the accumulation in renal tubular cells and the nephrotoxicity risk.
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IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.
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Antibacterianos , Infecções Urinárias , Animais , Camundongos , Testes de Sensibilidade Microbiana , Antibacterianos/uso terapêutico , Antibacterianos/farmacocinética , Tetraciclinas/farmacologia , Tetraciclinas/uso terapêutico , Infecções Urinárias/tratamento farmacológicoRESUMO
Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion.
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Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.
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BACKGROUND: Secondary metabolites have played a key role as starting points for drug development programs due to their often unique features compared with synthetically derived molecules. However, limitations related to the discovery and supply of these molecules by biotechnological means led to the retraction of big pharmaceutical companies from this field. The reasons included problems associated with strain culturing, screening, re-discovery, purification and characterization of novel molecules from natural sources. Nevertheless, recent reports have described technical developments that tackle such issues. While many of these reports focus on the identification and characterization of such molecules to enable subsequent chemical synthesis, a biotechnological supply strategy is rarely reported. This may be because production processes usually fall under proprietary research and/or few processes may meet the requirements of a pharmaceutical development campaign. We aimed to bridge this gap for illudin M-a fungal sesquiterpene used for the development of anticancer agents-with the intention to show that biotechnology can be a vital alternative to synthetic processes dealing with small molecules. RESULTS: We used µL-scale models to develop an adsorption and extraction strategy for illudin M recovery from culture supernatant of Omphalotus nidiformis and these findings were successfully transferred into lab-scale. By adsorbing and eluting the product using a fixed resin-bed we reduced the working volume by ~ 90% and removed the aqueous phase from the process. After a washing step, a highly concentrated illudin M fraction was obtained by isocratic elution with 80% methanol. The fraction was dried and extracted using a water/heptane mixture, enriching illudin M in the heptane phase. From heptane illudin M could be instantly crystalized by concentrating the solution, achieving a final purity > 95%. CONCLUSION: We have developed a robust, scalable and low-cost downstream process to obtain highly pure illudin M. By using solid phase extraction we reduced the production of solvent waste. Heptane from the final purification step could be recycled. The reduced amounts of solvents required, and the short purification time render this method a very economic and ecologic alternative to published processes.
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Antineoplásicos , Sesquiterpenos , Heptanos , Sesquiterpenos Policíclicos , Sesquiterpenos/química , SolventesRESUMO
BACKGROUND: The fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown improved in vitro selectivity towards cancer cells encouraging further investigation. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus. We have recently reported a robust shake flask process for the production of gram quantities of illudin M from Omphalotus nidiformis aiming to transfer that process into stirred tank bioreactors, which can be used in a commercial production set-up. However, process transfer across different systems is not straightforward and particularly challenging when the producer is morphologically complex. There are only a few reports that address the development of bioprocesses for the production of compounds from Basidiomycota as these organisms have not been extensively studied because of their complex life cycles and often are difficult to cultivate under laboratory conditions. RESULTS: The recently developed shake flask process delivering stable titers of ~ 940 mg L-1 of illudin M was investigated using off-gas analysis to identify critical parameters which facilitated the transfer from shaken into stirred tank bioreactors. Comparable titers to the shake flask process were achieved in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in 523 mg L-1 and is the starting point for optimization of the identified parameters at that scale. CONCLUSION: By identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into 2 L stirred tank bioreactors reaching a comparable titer (> 900 mg L-1), which is significantly higher than any previously reported. The insights obtained from 10 L scale pave the way towards further scale-up studies that will enable a sustainable supply of illudin M to support preclinical and clinical development programs.
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Reatores Biológicos , Sesquiterpenos Policíclicos , Biomassa , Reatores Biológicos/microbiologia , Sesquiterpenos Policíclicos/metabolismoRESUMO
Covering: August 1984 up to January 2022Worldwide, increasing morbidity and mortality due to antibiotic-resistant microbial infections has been observed. Therefore, better prevention and control of infectious diseases, as well as appropriate use of approved antibacterial drugs are crucial. There is also an urgent need for the continuous development and supply of novel antibiotics. Thus, identifying new antibiotics and their further development is once again a priority of natural product research. The antibiotic corallopyronin A was discovered in the 1980s in the culture broth of the Myxobacterium Corallococcus coralloides and serves, in the context of this review, as a show case for the development of a naturally occurring antibiotic compound. The review demonstrates how a hard to obtain, barely water soluble and unstable compound such as corallopyronin A can be developed making use of sophisticated production and formulation approaches. Corallopyronin A is a bacterial DNA-dependent RNA polymerase inhibitor with a new target site and one of the few representatives of this class currently in preclinical development. Efficacy against Gram-positive and Gram-negative pathogens, e.g., Chlamydia trachomatis, Orientia tsutsugamushi, Staphylococcus aureus, and Wolbachia has been demonstrated. Due to its highly effective in vivo depletion of Wolbachia, which are essential endobacteria of most filarial nematode species, and its robust macrofilaricidal efficacy, corallopyronin A was selected as a preclinical candidate for the treatment of human filarial infections. This review highlights the discovery and production optimization approaches for corallopyronin A, as well as, recent preclinical efficacy results demonstrating a robust macrofilaricidal effect of the anti-Wolbachia candidate, and the solid formulation strategy which enhances the stability as well as the bioavailability of corallopyronin A.
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Anti-Infecciosos , Produtos Biológicos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Produtos Biológicos/farmacologia , Humanos , Lactonas , ÁguaRESUMO
BACKGROUND: The fungal sesquiterpenes Illudin M and S are important base molecules for the development of new anticancer agents due to their strong activity against some resistant tumor cell lines. Due to nonspecific toxicity of the natural compounds, improvement of the pharmacophore is required. A semisynthetic derivative of illudin S (Irofulven) entered phase II clinical trials for the treatment of castration-resistant metastatic prostate cancer. Several semisynthetic illudin M derivatives showed increased in vitro selectivity and improved therapeutic index against certain tumor cell lines, encouraging further investigation. This requires a sustainable supply of the natural compound, which is produced by Basidiomycota of the genus Omphalotus. We aimed to develop a robust biotechnological process to deliver illudin M in quantities sufficient to support medicinal chemistry studies and future preclinical and clinical development. In this study, we report the initial steps towards this goal. RESULTS: After establishing analytical workflows, different culture media and commercially available Omphalotus strains were screened for the production of illudin M.Omphalotus nidiformis cultivated in a medium containing corn steep solids reached ~ 38 mg L-1 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L-1; corn steep solids 7.0 g L- 1; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L-1). Based on a reproducible cultivation method, a feeding strategy was developed considering potential biosynthetic bottlenecks. Acetate and glucose were fed at 96 h (8.0 g L-1) and 120 h (6.0 g L-1) respectively, which resulted in final illudin M titer of ~ 940 mg L-1 after eight days. This is a 25 fold increase compared to the initial titer. CONCLUSION: After strict standardization of seed-preparation and cultivation parameters, a combination of experimental design, empirical trials and additional supply of limiting biosynthetic precursors, led to a highly reproducible process in shake flasks with high titers of illudin M. These findings are the base for further work towards a scalable biotechnological process for a stable illudin M supply.
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Glucose , Linhagem Celular Tumoral , Sesquiterpenos PolicíclicosRESUMO
The rise of antimicrobial resistance poses a severe threat to public health. The natural product chlorotonil was identified as a new antibiotic targeting multidrug resistant Gram-positive pathogens and Plasmodium falciparum. Although chlorotonil shows promising activities, the scaffold is highly lipophilic and displays potential biological instabilities. Therefore, we strived towards improving its pharmaceutical properties by semisynthesis. We demonstrated stereoselective epoxidation of chlorotonils and epoxide ring opening in moderate to good yields providing derivatives with significantly enhanced solubility. Furthermore, in vivo stability of the derivatives was improved while retaining their nanomolar activity against critical human pathogens (e.g. methicillin-resistant Staphylococcus aureus and P. falciparum). Intriguingly, we showed further superb activity for the frontrunner molecule in a mouse model of S. aureus infection.
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Antimaláricos , Malária Falciparum , Staphylococcus aureus Resistente à Meticilina , Animais , Antibacterianos/farmacologia , Antimaláricos/farmacologia , Compostos de Epóxi/farmacologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Staphylococcus aureusRESUMO
Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.
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Produtos Biológicos/química , Lisina/análogos & derivados , Myxococcales/química , Niacina/química , Vias Biossintéticas/genética , Genoma Bacteriano/genética , Lisina/química , Metaboloma/genética , Myxococcales/genética , Myxococcales/isolamento & purificação , Niacina/isolamento & purificaçãoRESUMO
Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.
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Filariose/tratamento farmacológico , Filaricidas/uso terapêutico , Filarioidea/efeitos dos fármacos , Lactonas/uso terapêutico , Wolbachia/efeitos dos fármacos , Animais , Feminino , Filariose/parasitologia , Filarioidea/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Simbiose/efeitos dos fármacosRESUMO
The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.
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Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 µM and 4.97 µM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
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Antivirais/farmacologia , Bacteriocinas/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus Sincicial Respiratório Humano/fisiologiaRESUMO
To address the global challenge of emerging antimicrobial resistance, the hitherto most successful strategy to new antibiotics has been the optimization of validated natural products; most of these efforts rely on semisynthesis. Herein, we report the semisynthetic modification of amidochelocardin, an atypical tetracycline obtained via genetic engineering of the chelocardin producer strain. We report modifications at C4, C7, C10 and C11 by the application of methylation, acylation, electrophilic substitution, and oxidative C-C coupling reactions. The antibacterial activity of the reaction products was tested against a panel of Gram-positive and Gram-negative pathogens. The emerging structure-activity relationships (SARs) revealed that positions C7 and C10 are favorable anchor points for the semisynthesis of optimized derivatives. The observed SAR was different from that known for tetracyclines, which underlines the pronounced differences between the two compound classes.
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Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Tetraciclinas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tetraciclinas/síntese química , Tetraciclinas/químicaRESUMO
The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines (e.g. 16R-aminoratjadone 16 with IC50 = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC50 = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.
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The Basidiomycota constitutes the second largest higher taxonomic group of the Fungi after the Ascomycota and comprises over 30.000 species. Mycelial cultures of Basidiomycota have already been studied since the 1950s for production of antibiotics and other beneficial secondary metabolites. Despite the fact that unique and selective compounds like pleuromutilin were obtained early on, it took several decades more until they were subjected to a systematic screening for antimicrobial and anticancer activities. These efforts led to the discovery of the strobilurins and several hundreds of further compounds that mainly constitute terpenoids. In parallel the traditional medicinal mushrooms of Asia were also studied intensively for metabolite production, aimed at finding new therapeutic agents for treatment of various diseases including metabolic disorders and the central nervous system. While the evaluation of this organism group has in general been more tedious as compared to the Ascomycota, the chances to discover new metabolites and to develop them further to candidates for drugs, agrochemicals and other products for the Life Science industry have substantially increased over the past decade. This is owing to the revolutionary developments in -OMICS techniques, bioinformatics, analytical chemistry and biotechnological process technology, which are steadily being developed further. On the other hand, the new developments in polythetic fungal taxonomy now also allow a more concise selection of previously untapped organisms. The current review is dedicated to summarize the state of the art and to give an outlook to further developments.
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Basidiomycota , Agroquímicos , Ascomicetos , Ásia , Produtos BiológicosRESUMO
Total synthesis of cystobactamid 920-1 and its epimer has allowed an unambiguous assignment of the relative and absolute configuration of the natural product. A careful structural analysis of each isomer using both NMR and computational techniques also prompted a constitutional revision of the structures originally reported for cystobactamids 920-1 and 920-2, and has provided further insight into the unique conformational preferences of the cystobactamid family.
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A method was established for the production of 1.2-fold and 4.2-fold increased amounts of the antiviral and central nervous system-active lantipeptides, labyrinthopeptins A1 and A2, respectively, isolated from the actinobacterium Actinomadura namibiensis, to enable production in gram scale. We then performed in vivo characterization of this promising compound class. The labyrinthopeptins A1 and A2 have similar chemical structures and physical properties but differ drastically in their bioactivities. Therefore, large quantities of highly pure material are required for pharmacological studies. An effective methodology was established for the first time for their production in bioreactors, their separation involving gel permeation chromatography on LH20 material, followed by reversed phase-high performance liquid chromatography. With an optimized methodology, 580 mg of labyrinthopeptin A1 and 510 mg of labyrinthopeptin A2 were quantitatively isolated with recovery rates of 72.5% and 42.3% from 7.5 L of culture broth, respectively. However, the fermentation that had already resulted in maximum yields of over 100 mg/L of both target molecules after 300 h in a 10-L scale bioreactor, still requires further optimisation.
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Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cbâ vi35. Previously nothing had been reported about their biosynthesis, including the origin of the unusual 4-methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at the single-digit mg L-1 scale, we developed a stable host that eventually allowed for yields of up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S-adenosylmethionine (SAM)-dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, nonnatural vioprolide derivatives were generated via rational genetic engineering.
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Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxytoca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a nonenzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Finally, it could be shown that salicylic and acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably by blocking the peptidyl carrier protein ThdA, pointing toward a potential application in combination therapy to prevent or alleviate the symptoms of AAHC.