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Representative micrograph of mycelial aggregates found around the centroid of the right cluster, for each cultivation condition. a Standing culture; b–i MTP cultures shaken at b 600 rpm, c 800 rpm; d 1000 rpm; e 1200 rpm; f 1400 rpm; g 1600 rpm; h 1800 rpm; and i shake flask. Yellow perimeter signifies the measured object by image analysis. Scale bar: 100 µm and is shared for B-I. (Color figure online)
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Actinobacteria are prolific producers of secondary metabolites and industrially relevant enzymes. Growth of these mycelial micro-organisms in small culture volumes is challenging due to their complex morphology. Since morphology and production are typically linked, scaling down culture volumes requires better control over morphogenesis. In larger s...
Citations
... Dado que CoA2CA es demandante por la fase disipativa, dicho mecanismo abarcó el adosamiento a matrices abióticas de vidrio de borosilicato con el desarrollo de películas adherentes y facilitación para la dispersión bacteriana a nuevos espacios potencialmente colonizables ante recambios de fuentes hídricas (Young, 2006;Preedy et al., 2014). Aunque los argumentos previos pueden teorizar acertadamente los orígenes ecofuncionales del biocontrol, también es congruente con explicar la formación de gránulos esferoidales y arrosetados de CoA2CA, que podrían asociarse con la expresión de antibióticos e insecticidas de interés en bioprocesos tecnológicos (Dhakal et al., 2017;van Dissel y van Wezel, 2018), en vista que el morfotipo mostró mayor actividad larvicida. En ese sentido, la liberación de metabolitos larvicidas sería factible de ocurrir, si asumimos que se produzcan en las hifas de grumos y gránulos tal cual se realiza en otros actinomicetos a escala biotecnológica (Singh et al., 2012). ...
Actinobacteria are a group of widely known microorganisms used in the synthesis of insecticidal bioactive compounds. Nevertheless, over-exploitation of Streptomyces-derived metabolites has led to explore new bioactive molecules based on non-streptomycetes actinobacteria in order to minimize the development of insecticide resistance in Aedes aegypti. In accordance with the use of eco-friendly bioagents, in this study biofilm-forming actinobacteria were characterized on the basis of assessment their growth dynamics, larvicidal mortality and sublethal effects. Actinobacteria identification, biofilm growth and larvicidal bioactivities were performed by employing bacterial cultures, photomicrograph-based image analysis and bioassays. Results indicated that bacterial morphotypes belong to Pseudonocardiaceae (PsA1TA) and Corynebacteriaceae (CoA2CA), which showed a distinctly substrate-dependent growth. PsA1TA microcolonies were randomly distributed on abdominal and thoracic membranous epicuticle. Afterwards, the thickness of mono- and bi-layered biofilms were increased fourfold the larval thoracoabdominal width (infectious breadth, 1010 µm - 1036 µm). In contrast, cephalic and anal sclerotized structures were radially encased by CoA2CA biofilms and increased threefold the size of both structures (infectious breadth, 1820 - 2030 µm and 1650 - 1860 µm, respectively). Although biofilms caused dissimilar mortality rates on the four larval instars, PsA1TA exerted highest larvicidal activity and virulence on second instar larvae (58 %-96 hours, LT50: 3.4 days) and CoA2CA on fourth instar larvae (85 %-96 hours, LT50: 2.5 days). CoA2CA also induced incomplete release of pharate individuals as well as buckled protruding tarsi in newly emergent adults. Larval cadavers were overwhelmingly encased within massive biofilm aggregates. Biofilm-forming actinobacteria performed biolarvicidal activity and sublethal responses in A. aegypti.
... It has been suggested that the growth rate of adherent cells is enhanced when a certain cell density is reached, whereas the growth rate drops at higher densities. This density-dependent growth may be explained by cell-cell signaling, resulting in physical or morphological changes of the biofilm bacteria (Rajput et al., 2015;Betancur et al., 2017;van Dissel and van Wezel, 2018). Table 1 shows the composition of total protein, eDNA, and total EPS of biofilms samples, by Streptomyces sp. ...
The extracellular polymeric substances (EPS) have shown free radical scavenging and antitumor activity against both breast and colon cell lines. In this regard, actinobacteria have become an increasingly popular sources of EPS. Therefore, in this study four Streptomyces strains isolated from contaminated soil (M7, A5, A14 and MC1) were evaluated for determining its biofilm-forming capacity including under pesticide stress. In addition, chemical composition of EPS and its cytotoxic effects over 4T1 breast cancer cell and Caco-2 human tumor colon cells were evaluated. The results demonstrated that Streptomyces sp. A5 had the highest capability to develop biofilm more than other strains tested, even under pesticide stress. Moreover, this strain produced EPS with a total protein/total polysaccharide rate of 1.59 ± 0.05. On the other hand, cytotoxicity assays of EPS showed that Streptomyces sp. A5 display a higher toxic effect against 4T1 Breast cancer cells (96.2 ± 13.5 %), Caco-2 (73.9 ± 6.4 %) and low toxicity (29.9 % ± 9.1 %) against non-transformed intestinal cells (IEC-18). Data do not show cytotoxic effect relationship with biofilm-forming capabilities of strains, nor the chemical composition of EPS matrix. The gene that codes for polysaccharide deacetylase, parB-like and transRDD proteins, were identified. These results contribute to the knowledge about the variability of chemical composition and potential cytotoxic properties of EPS produced by Streptomyces biofilms. It proposes interesting future challenges for linking Streptomyces-based pesticide remediation technology with the development of new antitumor drugs.
... As shown, talc reduced the pellet size of both strains (Figure 3a) , and this response can be expected also in other actinobacteria (Ren et al., 2015), although exceptions cannot be excluded. From the bioengineering viewpoint, smaller pellets appear favorable, as they reduce problems with mass and oxygen transfer limitation, slow growth, and culture heterogeneity (Mehmood et al., 2012;Tamura et al., 1997;van Dissel & van Wezel, 2018;van Dissel et al., 2014). ...
Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2·H2O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met‐bottromycin A2 up to 109 mg L⁻¹, the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L⁻¹ talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re‐balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non‐cyclized shunt by‐products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13‐fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline‐type oxytetracycline, and the anthramycin‐analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone‐type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle‐supplementation appears as an enabling technology in natural product research of these most important microbes.
... During this work, we observed a strong size reduction of cell pellets in microparticle presence and gathered detailed information on how the talc microparticles modified and accelerated morphogenesis in submerged cultures of S. albus and S. lividans (Kuhl et al., 2020;Kuhl et al., 2021). It appears clear that smaller pellet diameters are favored from a bioprocess engineering point of view, as there are less problems with mass transfer rates, slow growth, and culture heterogeneity in liquid cultures (Tamura et al., 1997;Mehmood et al., 2012;van Dissel et al., 2014;van Dissel & van Wezel, 2018;Kuhl et al., 2021). In the cases presented here, talc addition resulted in a 40% size reduction of submerged S. lividans pellets during growth and even a more than sixfold size reduction for pellets of pamamycin producing S. albus (Kuhl et al., 2020;Kuhl et al., 2021 (Zacchetti et al., 2018). ...
... Moreover, it appears interesting to investigate the addition of microparticles to actinobacterial strains, which are, at least for now, genetically not accessible (Atanasov et al., 2021). In this regard, the approach is also an excellent addition to recently developed approaches for the micro-scale characterization of Streptomyces (Koepff et al., 2017;van Dissel & van Wezel, 2018). ...
... These parameters must be considered when scaling up production to industrial conditions [65]. Interestingly, a recent study downscaled liquid cultures to the 100 µL scale in microtiter plates [66], reproducing the same range of production and morphology as large-scale bioreactors, making screening easier and facilitating further upscaling. ...
Streptomyces is a diverse group of gram-positive microorganisms characterised by a complex developmental cycle. Streptomycetes produce a number of antibiotics and other bioactive compounds used in the clinic. Most screening campaigns looking for new bioactive molecules from actinomycetes have been performed empirically, e.g., without considering whether the bacteria are growing under the best developmental conditions for secondary metabolite production. These screening campaigns were extremely productive and discovered a number of new bioactive compounds during the so-called "golden age of antibiotics" (until the 1980s). However, at present, there is a worrying bottleneck in drug discovery, and new experimental approaches are needed to improve the screening of natural actinomycetes. Streptomycetes are still the most important natural source of antibiotics and other bioactive compounds. They harbour many cryptic secondary metabolite pathways not expressed under classical laboratory cultures. Here, we review the new strategies that are being explored to overcome current challenges in drug discovery. In particular, we focus on those aimed at improving the differentiation of the antibiotic-producing mycelium stage in the laboratory.
Antibiotic-resistant illnesses are on the rise worldwide, and the pipeline for developing new antibiotics is drying up. As a result, researchers need to create novel compounds with antimicrobial action. Recent decades have seen a dearth of novel antibiotics because of the reliance on conventional empirical screening procedures using both natural and synthetic chemicals to find them. There is hope that the massive amount of bacterial genome sequence data that has become accessible since the sequencing of the first bacterial genome more than 20 years ago might help lead to the development of new antibiotic drugs. Genes with significant levels of conservation both within and between bacterial species can be found using comparative genomic techniques; these genes may be involved in essential bacterial functions. Bioactive chemicals found in natural products have been successfully used in treating everything from infectious diseases to cancer, but over the past 20-30 years, the effectiveness of screening methods based on fermentation has decreased. Researchers urgently need answers to the unmet demand for bacterial infection resistance. Now more than ever, with the advent of cheap, high-throughput genomic sequencing technology, natural product discovery can be revitalized. Using bioinformatics, investigators may foretell whether or not a certain microbial strain would generate compounds with novel chemical structures, which may have novel modes of action in inhibiting bacterial growth. This manuscript describes how this potential might be utilised, with a particular emphasis on manipulating the expression of dormant biosynthetic gene clusters that are hypothesised to encode new antibiotics. Additionally, it consolidates the work of the past and the present to utilise bacterial genomic data in the identification and development of new antibiotics.
Streptomyces genera serve as adaptable cell factories for secondary metabolites with various and distinctive chemical structures that are relevant to the pharmaceutical industry. Streptomyces' complex life cycle necessitated a variety of tactics to enhance metabolite production. Identification of metabolic pathways, secondary metabolite clusters, and their controls have all been accomplished using genomic methods. Besides this, bioprocess parameters were also optimized for the regulation of morphology. Kinase families were identified as key checkpoints in the metabolic manipulation (DivIVA, Scy, FilP, matAB, and AfsK) and morphology engineering of Streptomyces. This review illustrates the role of different physiological variables during fermentation in the bioeconomy coupled with genome-based molecular characterization of biomolecules responsible for secondary metabolite production at different developmental stages of the Streptomyces life cycle.
Natural products are a rich source of bioactive compounds that have been used successfully in the areas of human health from infectious disease to cancer; however, traditional fermentation-based screening has provided diminishing returns over the last 20-30 years. Solutions to the unmet need of resistant bacterial infection are critically required. Technological advances in high-throughput genomic sequencing, coupled with ever-decreasing cost, are now presenting a unique opportunity for the reinvigoration of natural product discovery. Bioinformatic methods can predict the propensity of a microbial strain to produce molecules with novel chemical structures that could have new mechanisms of action in bacterial growth inhibition. This review highlights how this potential can be harnessed; with a focus on engineering the expression of silent biosynthetic gene clusters predicted to encode novel antibiotics.
Streptomycetes are multicellular filamentous microorganisms, and major producers of industrial enzymes and bioactive compounds such as antibiotics and anticancer drugs. The mycelial lifestyle plays an important role in the productivity during industrial fermentations. The hyphae of liquid-grown streptomycetes can self-aggregate into pellets, which hampers their industrial exploitation. Here we show that the Mat complex, which is required for pellet formation, catalyzes the synthesis of extracellular poly-β-1,6-N-acetylglucosamine (PNAG) in the model organisms Streptomyces coelicolor and Streptomyces lividans. Extracellular accumulation of PNAG allows Streptomyces to attach to hydrophilic surfaces, while attachment to hydrophobic surfaces requires a cellulase-degradable extracellular polymer (EPS) produced by CslA. Over-expression of matAB was sufficient to restore pellet formation to cslA null mutants of S. lividans. The two EPS systems together increase the robustness of mycelial pellets. These new insights allow better control of liquid-culture morphology of streptomycetes, which may be harnessed to improve growth and industrial exploitation of these highly versatile natural product and enzyme producers.
