-
Frontiers in Bioengineering and... 2023In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular...
In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.
PubMed: 37731767
DOI: 10.3389/fbioe.2023.1254136 -
Frontiers in Bioengineering and... 2023, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is...
Morphology engineering for novel antibiotics: Effect of glass microparticles and soy lecithin on rebeccamycin production and cellular morphology of filamentous actinomycete .
, as an actinomycete, is a natural producer of the antibiotic and antitumoral drug rebeccamycin. Due to the filamentous cellular morphology handling in cultivations is challenging; therefore, morphology engineering techniques are mandatory to enhance productivity. One promising approach described in the literature is the addition of mineral particles in the micrometer range to precisely adjust cellular morphology and the corresponding product synthesis (microparticle-enhanced cultivation, MPEC). Glass microparticles are introduced in this study as a novel supplementation type for bioprocess intensification in filamentous organisms. Several investigations were conducted to screen for an optimal particle setup, including particle size and concentration regarding their impact and effects on enhanced productivity, microparticle incorporation behavior into the biopellets, the viability of pellets, and morphological changes. Glass microparticles (10 g·L) with a median diameter of 7.9 µm, for instance, induced an up to fourfold increase in product synthesis accompanied by overall enhanced viability of biomass. Furthermore, structural elucidations showed that biopellets isolated from MPEC tend to have lower hyphal density than unsupplemented control pellets. In this context, oxygen microprofiling was conducted to better understand how internal structural changes interwind with oxygen supply into the pellets. Here, the resulting oxygen profiles are of a contradictive trend of steeper oxygen consumption with increasing glass microparticle supplementation. Eventually, MPEC was combined with another promising cultivation strategy, the supplementation of soy lecithin (7.5 g·L), to further increase the cultivation performance. A combination of both techniques in an optimized setup resulted in a rebeccamycin concentration of 213 mg·L after 10 days of cultivation, the highest value published so far for microparticle-supplemented shake flask cultivations of .
PubMed: 37091334
DOI: 10.3389/fbioe.2023.1171055 -
Molecules (Basel, Switzerland) Oct 2022The present study is to explore the anticancer effect of loonamycin (LM) in vitro and in vivo, and investigate the underlying mechanism with combined multi-omics. LM...
The present study is to explore the anticancer effect of loonamycin (LM) in vitro and in vivo, and investigate the underlying mechanism with combined multi-omics. LM exhibited anticancer activity in human triple negative breast cancer cells by promoting cell apoptosis. LM administration inhibited the growth of MDA-MB-468 tumors in a murine xenograft model of breast cancer. Mechanistic studies suggested that LM could inhibit the topoisomerase I in a dose-dependent manner in vitro experiments. Combined with the transcriptomics and proteomic analysis, LM has a significant effect on O-glycan, p53-related signal pathway and EGFR/PI3K/AKT/mTOR signal pathway in enrichment of the KEGG pathway. The GSEA data also suggests that the TNBC cells treated with LM may be regulated by p53, O-glycan and EGFR/PI3K/AKT/mTOR signaling pathway. Taken together, our findings predicted that LM may target p53 and EGFR/PI3K/AKT/mTOR signaling pathway, inhibiting topoisomerase to exhibit its anticancer effect.
Topics: Humans; Mice; Animals; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; DNA Topoisomerases, Type I; Tumor Suppressor Protein p53; Transcriptome; Proteomics; Cell Line, Tumor; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Apoptosis; ErbB Receptors; Cell Proliferation
PubMed: 36296549
DOI: 10.3390/molecules27206958 -
Metabolites Mar 2022Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In...
Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In and , regulation of virulence genes' expression occurs through the cyclic peptide-mediated accessory gene regulator () and its ortholog quorum sensing systems, respectively. In the present study, we screened a set of 54 actinomycetales secondary metabolites as novel anti-virulence compounds targeting quorum sensing system of the Gram-positive bacteria. The results indicated that four compounds, Phenalinolactones A-D, BU-4664LMe, 4,5-dehydrogeldamycin, and Questinomycin A, potentially inhibit the quorum sensing system and hemolytic activity of . On the other hand, Decatromicin A and B, Okilactomycin, Rishirilide A, Abyssomicin I, and Rebeccamycin selectively blocked the quorum sensing system and the gelatinase production in at sub-lethal concentrations. Interestingly, Synerazol uniquely showed the capability to inhibit both and quorum sensing systems. Further, in silico molecular docking studies were performed which provided closer insights into the mode of action of these compounds and proposed that the inhibitory activity of these compounds could be attributed to their potential ability to bind to the ATP-active site of AgrA. Taken together, our study highlights the potential of actinomycetales secondary metabolites with diverse structures as anti-virulence quorum sensing inhibitors.
PubMed: 35323689
DOI: 10.3390/metabo12030246 -
Molecules (Basel, Switzerland) Dec 2021Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied...
Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied the mechanisms of anticancer activity in accordance with the previously described effects of indolocarbazoles. Cytotoxicity was estimated by MTT assay. We analyzed LCS-DNA interactions by circular dichroism in cholesteric liquid crystals and fluorescent indicator displacement assay. The effect on the activity of topoisomerases I and II was studied by DNA relaxation assay. Expression of interferon signaling target genes was estimated by RT-PCR. Chromatin remodeling was analyzed-the effect on histone H1 localization and reactivation of epigenetically silenced genes. LCS-induced change in the expression of a wide gene set was counted by means of PCR array. Our study revealed the cytotoxic activity of the compounds against 11 cancer cell lines and it was higher than in immortalized cells. Both compounds bind DNA; binding constants were estimated-LCS-1208 demonstrated higher affinity than LCS-1269; it was shown that LCS-1208 intercalates into DNA that is typical for rebeccamycin derivatives. LCS-1208 also inhibits topoisomerases I and IIα. Being a strong intercalator and topoisomerase inhibitor, LCS-1208 upregulates the expression of interferon-induced genes. In view of LCSs binding to DNA we analyzed their influence on chromatin stability and revealed that LCS-1269 displaces histone H1. Our analysis of chromatin remodeling also included a wide set of epigenetic experiments in which LCS-1269 demonstrated complex epigenetic activity. Finally, we revealed that the antitumor effect of the compounds is based not only on binding to DNA and chromatin remodeling but also on alternative mechanisms. Both compounds induce expression changes in genes involved in neoplastic transformation and target genes of the signaling pathways in cancer cells. Despite of being structurally similar, each compound has unique biological activities. The effects of LCS-1208 are associated with intercalation. The mechanisms of LCS-1269 include influence on higher levels such as chromatin remodeling and epigenetic effects.
Topics: Antineoplastic Agents; Carbazoles; Cell Line, Tumor; Epigenesis, Genetic; Glycosides; Humans; Indoles; Neoplasms
PubMed: 34885910
DOI: 10.3390/molecules26237329 -
PloS One 2020Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process...
Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 μm, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 μm without and 106 μm with glass beads, diameters from flow cytometry were 170 and 100 μm, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability.
Topics: Actinomycetales; Carbazoles; Chromatography, High Pressure Liquid; Flow Cytometry; Image Processing, Computer-Assisted; Microscopy; Microscopy, Confocal
PubMed: 32492063
DOI: 10.1371/journal.pone.0234125 -
Biomolecules Apr 2020Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of and genera that affect poor and remote populations in developing...
Chagas disease and leishmaniasis are neglected tropical diseases caused by kinetoplastid parasites of and genera that affect poor and remote populations in developing countries. These parasites share similar complex life cycles and modes of infection. It has been demonstrated that the particular group of phosphorylating enzymes, protein kinases (PKs), are essential for the infective mechanisms and for parasite survival. The natural indolocarbazole staurosporine (STS, ) has been extensively used as a PKC inhibitor and its antiparasitic effects described. In this research, we analyze the antikinetoplastid activities of three indolocarbazole (ICZs) alkaloids of the family of staurosporine STS, -, and the commercial ICZs rebeccamycin (), K252a (), K252b (), K252c (), and arcyriaflavin A () in order to establish a plausive approach to the mode of action and to provide a preliminary qualitative structure-activity analysis. The most active compound was 7-oxostaurosporine (7OSTS, ) that showed IC values of 3.58 ± 1.10; 0.56 ± 0.06 and 1.58 ± 0.52 µM against and and a Selectivity Index (CC/IC) of 52 against amastigotes of compared to the J774A.1 cell line of mouse macrophages.
Topics: Animals; Antiprotozoal Agents; Biological Assay; Carbazoles; Cell Death; Cell Line; Inhibitory Concentration 50; Kinetoplastida; Membrane Potential, Mitochondrial; Metabolome; Mice; Mitochondria; Models, Molecular; Parasites; Staurosporine; Streptomyces; Toxicity Tests
PubMed: 32344693
DOI: 10.3390/biom10040657 -
ACS Omega May 2018Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and...
Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenases-PrnA and PyrH-was investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site.
PubMed: 31458701
DOI: 10.1021/acsomega.8b00385 -
Journal of the American Chemical Society Jan 2018Extensive effort has been devoted to engineering flavin-dependent halogenases (FDHs) with improved stability, expanded substrate scope, and altered regioselectivity....
Extensive effort has been devoted to engineering flavin-dependent halogenases (FDHs) with improved stability, expanded substrate scope, and altered regioselectivity. Here, we show that variants of rebeccamycin halogenase (RebH) catalyze enantioselective desymmetrization of methylenedianilines via halogenation of these substrates distal to their pro-stereogenic center. Structure-guided engineering was used to increase the conversion and selectivity of these reactions, and the synthetic utility of the halogenated products was shown via conversion of to a chiral α-substituted indole. These results constitute the first reported examples of asymmetric catalysis by FDHs.
Topics: Aniline Compounds; Catalysis; Flavins; Halogenation; Methylene Chloride; Molecular Structure; Protein Engineering; Stereoisomerism
PubMed: 29294291
DOI: 10.1021/jacs.7b09573 -
Cellular Physiology and Biochemistry :... 2017Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment...
BACKGROUND/AIMS
Although proinflammatory cytokine-induced disruption of intestinal epithelial barrier integrity is associated with intestinal inflammatory disease, effective treatment for barrier dysfunction is lacking. Previously, we demonstrated that rebeccamycin alleviates epithelial barrier dysfunction induced by inflammatory cytokines in Caco-2 cell monolayers; however, the underlying mechanism remained unclear. Here, we investigated the mechanism by which rebeccamycin protects the epithelial barrier function of Caco-2 cells exposed to TNF-α.
METHODS
To confirm the epithelial barrier function of Caco-2 cell monolayers, transepithelial electrical resistance (TER) and paracellular permeability were measured. Production levels and localization of tight junction (TJ) proteins were analyzed by immunoblot and immunofluorescence, respectively. Phosphorylated myosin light chain (pMLC) and MLC kinase (MLCK) mRNA expression levels were determined by immunoblot and quantitative RT-PCR, respectively.
RESULTS
Rebeccamycin attenuated the TNF-α-induced reduction in TER and increase in paracellular permeability. Rebeccamycin increased claudin-5 expression, but not claudin-1, -2, -4, occludin or ZO-1 expression, and prevented the TNF-α-induced changes in ZO-1 and occludin localization. Rebeccamycin suppressed the TNF-α-induced increase in MLCK mRNA expression, thus suppressing MLC phosphorylation. The rebeccamycin-mediated reduction in MLCK production and protection of epithelial barrier function were alleviated by Chk1 inhibition.
CONCLUSION
Rebeccamycin attenuates TNF-α-induced disruption of intestinal epithelial barrier integrity by inducing claudin-5 expression and suppressing MLCK production via Chk1 activation.
Topics: Caco-2 Cells; Carbazoles; Checkpoint Kinase 1; Claudin-5; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Intestinal Mucosa; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Tight Junctions; Tumor Necrosis Factor-alpha
PubMed: 28391269
DOI: 10.1159/000472367