The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Sang Yup Lee - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 crispri and crispr best mediated genetic manipulation in Streptomycetes
Nature Protocols, 2020Co-Authors: Yaojun Tong, Christopher M Whitford, Kai Blin, Tue Sparholt Jorgensen, Tilmann Weber, Sang Yup LeeAbstract:Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for Streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target Streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.
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toward systems metabolic engineering of Streptomycetes for secondary metabolites production
Biotechnology Journal, 2018Co-Authors: Helene Lunde Robertsen, Tilmann Weber, Hyun Uk Kim, Sang Yup LeeAbstract:Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of Streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism. This new trend has been accompanied with development of new computational resources (genome and compound mining tools), generation of various high-quality omics data, establishment of molecular tools, and other strain engineering strategies. They all come together to enable systems metabolic engineering of Streptomycetes, allowing more systematic and efficient strain development. In this review, the authors present recent progresses within systems metabolic engineering of Streptomycetes for uncovering their hidden potential to produce novel compounds and for the improved production of secondary metabolites.
Linda L Kinkel - One of the best experts on this subject based on the ideXlab platform.
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plant community effects on the diversity and pathogen suppressive activity of soil Streptomycetes
Applied Soil Ecology, 2010Co-Authors: Matthew G Bakker, Jerry D Glover, John G Mai, Linda L KinkelAbstract:Ecological factors that promote pathogen suppressive microbial communities remain poorly understood. However, plants have profound impacts on the structure and functional activities of soil microbial communities, and land-use changes which alter plant diversity or community composition may indirectly affect the structure and function of microbial communities. Previous research has suggested that the Streptomycetes are significant contributors to pathogen suppression in soils. We compared soil streptomycete communities from high and low plant diversity treatments using an experimental manipulation that altered plant diversity while controlling for soil structure and disturbance. Specifically, we characterized an isolate collection for inhibition of plant pathogens as a measure of functional activity, and for 16S rDNA sequence to measure community structure. In this system, high and low diversity plant communities supported streptomycete communities with similar diversity, phylogenetic composition, and pathogen suppressive activity. However, inhibitory phenotypes differed among treatments for several phylogenetic groups, indicating that local selection is leading to divergence between Streptomycetes from high and low plant diversity communities. Although the ability to inhibit plant pathogens was common among soil Streptomycetes, pathogen-inhibitory activity differed widely among phylogenetic groups. The breadth and intensity of pathogen inhibition by soil Streptomycetes were positively related.
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management of soil microbial communities to enhance populations of fusarium graminearum antagonists in soil
Plant and Soil, 2008Co-Authors: C A Perez, Ruth Dillmacky, Linda L KinkelAbstract:Fusarium head blight (FHB), incited by Fusarium graminearum Schwabe is one of the most devastating diseases of wheat. Primary inoculum generated on crop residue is the driving force of FHB epidemics. Fusarium survival on crop residues is affected by soil microbial antagonists. The incorporation of green manures has been shown to increase the density and diversity of microbes in soils, particularly the density and the pathogen-inhibitory activity of specific bacteria and fungi. Evidence of increased streptomycete populations in soil as a response to green manure incorporation, and their negative effect on the survival of Fusarium oxysporum Schlechtendahl in soil, suggests their potential use to reduce the survival of related pathogens. There is, however, no precedent for the use of green manures to promote indigenous streptomycete populations to control FHB. This study investigated the use of green manures (sorghum–sudangrass hybrid [Sorghum bicolor (L.) Moench–S. bicolor (L.) Moench var. sudanense (Piper)] and common buckwheat [Fagopyrum esculentum (Moench)]) for reducing F. graminearum survival in association with wheat residues. Soil bacterial density, streptomycete density and the density and inhibitory activity of F. graminearum-antagonists were monitored from planting until 3 and 6 months following the incorporation of green manures in greenhouse and field experiments, respectively. The decomposition of wheat residues and survival of Fusarium in residues was also assessed. The use of green manures did not statistically impact the survival of F. graminearum in wheat residue. However, green manures promoted the development of higher densities and antagonistic abilities of F. graminearum-antagonists in soils. Additionally, streptomycete densities and F. graminearum-antagonist densities were significantly and positively correlated with reduced survival of Fusarium. The results of our study suggest that the use of green manures can enhance populations of indigenous soil microorganisms antagonistic to the survival of F. graminearum in wheat residue.
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green manures and crop sequences influence potato diseases and pathogen inhibitory activity of indigenous Streptomycetes
Phytopathology, 2005Co-Authors: B E Wiggins, Linda L KinkelAbstract:ABSTRACT A 2-year field trial was conducted to determine the effects of green manures and crop sequences on potato scab and Verticillium wilt. In addition, indigenous streptomycete densities and in vitro pathogen inhibitory activity were measured and their relationships to plant disease were determined. Green manures (buckwheat, canola, or fallow controls) were tested in conjunction with three crop sequences (alfalfa-potato, cornpotato, and potato-potato). Compared with fallow controls, tubers grown in buckwheat-treated soil had significantly lower Verticillium wilt ratings, and tubers grown in buckwheat- or canola-treated soil had greater yields. Potatoes grown in soil planted to corn or alfalfa the previous year had significantly lower Verticillium wilt and potato scab ratings as well as higher yields than potatoes grown in soil previously planted to potato. Streptomycetes from soils collected from green manure-treated plots tended to have greater in vitro pathogen inhibitory activity than streptomycete...
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green manures and crop sequences influence alfalfa root rot and pathogen inhibitory activity among soil borne Streptomycetes
Plant and Soil, 2005Co-Authors: Elizabeth B Wiggins, Linda L KinkelAbstract:A two-year trial was conducted to determine the effects of green manures and crop sequences on plant disease, streptomycete and bacterial densities, and inhibitory activity of indigenous Streptomycetes against four target pathogens. Green manure treatments, buckwheat (Fagopyrum esculentum L.), canola (Brassica napus L.), sorghum-sudangrass (Sorghum bicolor) (L.) Moench × Sorghum sudanense (Piper) Stapf.), and fallow control were tested in conjunction with three crop sequences in a Phytophthora-infested soil placed in containers. Alfalfa (Medicago sativa L.), potato (Solanum tubersoum L.), or corn (Zea mays L.) was grown in the first year, and alfalfa was grown in all containers in the second year. Compared to fallow controls, alfalfa grown in sorghum-sudangrass- or buckwheat-treated soil had significantly greater stand counts and total biomass, respectively. In addition, alfalfa grown in fallow-treated soils had the greatest Phytophthora root rot as a function of stand count. Crop rotation also had a significant effect on alfalfa root rot and yield. Potato scab disease intensity was greatest on tubers grown in fallow-treated soils, while tubers grown in canola-treated soils had the highest yields (total tuber weight). Green-manure-treated soils tended to have greater streptomycete and bacterial densities than fallow-treated soils. In addition, buckwheat- or sorghum-sudangrass-treated soils had greater proportions of Streptomycetes that were antagonistic against the target pathogens than fallow-treated soils. The proportion of antagonists in soil was negatively correlated with alfalfa root rot, and positively correlated with alfalfa stand counts. Inhibitory activity of the Streptomycetes was also negatively correlated with potato scab and positively correlated with potato yield. These data suggest that green manures may provide a strategy for increasing pathogen inhibitory activity within the streptomycete community in soil, and, in conjunction with crop rotation, may contribute to the control of a diverse collection of soil-borne plant pathogens on multiple crop species.
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genetic and phenotypic traits of Streptomycetes used to characterize antibiotic activities of field collected microbes
Canadian Journal of Microbiology, 2004Co-Authors: Anita L Davelos, Kun Xiao, Jennifer M Flor, Linda L KinkelAbstract:Although antibiotic production may contribute significantly to microbial fitness, there is limited information on the ecology of antibiotic-producing microbial populations in soil. Indeed, quantitative information on the variation in frequency and intensity of specific antibiotic inhibitory and resistance abilities within soil microbial communities is lacking. Among the Streptomycetes, antibiotic production is highly variable and resistance to antibiotics is highly specific to individual microbial strains. The objective of this work was to genetically and phenotypically characterize a reference collection of Streptomycetes for use in distinguishing inhibition and resistance phenotypes of field-collected microbes. Specifically, we examined inhibition and resistance abilities of all isolates in all possible pairwise combinations, genetic relatedness using BOX-PCR and 16S rDNA sequence analyses, nutrient utilization profiles, and antibiotic induction among all possible three-way combinations of isolates. Eac...
Gilles P Van Wezel - One of the best experts on this subject based on the ideXlab platform.
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Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production
BMC, 2018Co-Authors: Boris Zacchetti, Gilles P Van Wezel, Agathoklis Andrianos, Dino Van Dissel, Evelien De Ruiter, Dennis ClaessenAbstract:Abstract Background Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many Streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics. Results Here, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth. Conclusions Our data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of Streptomycetes
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micro encapsulation extends mycelial viability of streptomyces lividans 66 and increases enzyme production
bioRxiv, 2017Co-Authors: Boris Zacchetti, Gilles P Van Wezel, Agathoklis Andrianos, Dino Van Dissel, Evelien De Ruiter, Dennis ClaessenAbstract:Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many Streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics. Here, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth. Our data demonstrate the utility of microencapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of Streptomycetes.
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intertwining nutrient sensory networks and the control of antibiotic production in streptomyces
Molecular Microbiology, 2016Co-Authors: Mia Urem, Magdalena A świątekpolatynska, Sebastien Rigali, Gilles P Van WezelAbstract:Actinobacteria are producers of a plethora of natural products of agricultural, biotechnological and clinical importance. In an era where mankind has to deal with rapidly spreading antimicrobial resistance, Streptomycetes are of particular importance as producers of half of all antibiotics used in the clinic. Genome sequencing efforts revealed that their capacity as antibiotic producers has been underestimated, in particular as many biosynthetic pathways are silent under standard laboratory conditions. Here we review the global regulatory networks that control antibiotic production in Streptomycetes, with emphasis on carbon- and aminosugar-related nutrient sensory pathways. Recent research has revealed intriguing connections between these regulons, and overlap and antagonism between the activities of among others the global regulatory proteins AtrA, DasR and Rok7B7 as well as GlnR (nitrogen control) and PhoP (phosphate control), are discussed. Finally, we provide ideas as to how these novel insights might help us to find ways to activate the transcription of silent biosynthetic gene clusters.
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unsuspected control of siderophore production by n acetylglucosamine in Streptomycetes
Environmental Microbiology Reports, 2012Co-Authors: Matthias Craig, Gilles P Van Wezel, Severine Colson, Stephany Lambert, Samuel Jourdan, Elodie Tenconi, Marta Maciejewska, Marc Ongena, Juan Francisco Martin, Sebastien RigaliAbstract:Summary Iron is one of the most abundant elements on earth but is found in poorly soluble forms hardly accessible to microorganisms. To subsist, they have developed iron-chelating molecules called siderophores that capture this element in the environment and the resulting complexes are internalized by specific uptake systems. While biosynthesis of siderophores in many bacteria is regulated by iron availability and oxidative stress, we describe here a new type of regulation of siderophore production. We show that in Streptomyces coelicolor, their production is also controlled by N-acetylglucosamine (GlcNAc) via the direct transcriptional repression of the iron utilization repressor dmdR1 by DasR, the GlcNAc utilization regulator. This regulatory nutrient–metal relationship is conserved among Streptomycetes, which indicates that the link between GlcNAc utilization and iron uptake repression, however unsuspected, is the consequence of a successful evolutionary process. We describe here the molecular basis of a novel inhibitory mechanism of siderophore production that is independent of iron availability. We speculate that the regulatory connection between GlcNAc and siderophores might be associated with the competition for iron between Streptomycetes and their fungal soil competitors, whose cell walls are built from the GlcNAc-containing polymer chitin. Alternatively, GlcNAc could emanate from Streptomycetes’ own peptidoglycan that goes through intense remodelling throughout their life cycle, thereby modulating the iron supply according to specific needs at different stages of their developmental programme.
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cell division and dna segregation in streptomyces how to build a septum in the middle of nowhere
Molecular Microbiology, 2012Co-Authors: Dagmara Jakimowicz, Gilles P Van WezelAbstract:Summary Streptomycetes are antibiotic-producing filamentous microorganisms that have a mycelial life style. In many ways Streptomycetes are the odd ones out in terms of cell division. While the basic components of the cell division machinery are similar to those found in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, many aspects of the control of cell division and its co-ordination with chromosome segregation are remarkably different. The rather astonishing fact that cell division is not essential for growth makes these bacteria unique. The fundamental difference between the cross-walls produced during normal growth and sporulation septa formed in aerial hyphae, and the role of the divisome in their formation are discussed. We then take a closer look at the way septum site localization is regulated in the long and multinucleoid Streptomyces hyphae, with particular focus on actinomycete-specific proteins and the role of nucleoid segregation and condensation.
Tilmann Weber - One of the best experts on this subject based on the ideXlab platform.
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the design build test learn cycle for metabolic engineering of Streptomycetes
Essays in Biochemistry, 2021Co-Authors: Christopher M Whitford, Pablo Cruzmorales, Jay D Keasling, Tilmann WeberAbstract:Streptomycetes are producers of a wide range of specialized metabolites of great medicinal and industrial importance, such as antibiotics, antifungals, or pesticides. Having been the drivers of the golden age of antibiotics in the 1950s and 1960s, technological advancements over the last two decades have revealed that very little of their biosynthetic potential has been exploited so far. Given the great need for new antibiotics due to the emerging antimicrobial resistance crisis, as well as the urgent need for sustainable biobased production of complex molecules, there is a great renewed interest in exploring and engineering the biosynthetic potential of Streptomycetes. Here, we describe the Design-Build-Test-Learn (DBTL) cycle for metabolic engineering experiments in Streptomycetes and how it can be used for the discovery and production of novel specialized metabolites.
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crispr cas9 crispri and crispr best mediated genetic manipulation in Streptomycetes
Nature Protocols, 2020Co-Authors: Yaojun Tong, Christopher M Whitford, Kai Blin, Tue Sparholt Jorgensen, Tilmann Weber, Sang Yup LeeAbstract:Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for Streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target Streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.
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toward systems metabolic engineering of Streptomycetes for secondary metabolites production
Biotechnology Journal, 2018Co-Authors: Helene Lunde Robertsen, Tilmann Weber, Hyun Uk Kim, Sang Yup LeeAbstract:Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of Streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism. This new trend has been accompanied with development of new computational resources (genome and compound mining tools), generation of various high-quality omics data, establishment of molecular tools, and other strain engineering strategies. They all come together to enable systems metabolic engineering of Streptomycetes, allowing more systematic and efficient strain development. In this review, the authors present recent progresses within systems metabolic engineering of Streptomycetes for uncovering their hidden potential to produce novel compounds and for the improved production of secondary metabolites.
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exploiting the genetic potential of polyketide producing Streptomycetes
Journal of Biotechnology, 2003Co-Authors: Tilmann Weber, Katrin Welzel, Stefan Pelzer, Andreas Vente, Wolfgang WohllebenAbstract:Streptomycetes are the most important bacterial producers of bioactive secondary metabolites such as antibiotics or cytostatics. Due to the emerging resistance of pathogenic bacteria to all commonly used antibiotics, new and modified natural compounds are required for the development of novel drugs. In addition to the classical screening for natural compounds, genome driven approaches like combinatorial biosynthesis are permanently gaining relevance for the generation of new structures. This technology utilizes the combination of genes from different biosynthesis pathways resulting in the production of novel or modified metabolites. The basis for this strategy is the access to a significant number of genes and the knowledge about the activity and specificity of the enzymes encoded by them. A joint initiative was started to exploit the biosynthesis gene clusters from Streptomycetes. In this publication, an overview of the strategy for the identification and characterization of numerous biosynthesis gene clusters for polyketides displaying interesting functions and particular structural features is given.
B E Wiggins - One of the best experts on this subject based on the ideXlab platform.
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green manures and crop sequences influence potato diseases and pathogen inhibitory activity of indigenous Streptomycetes
Phytopathology, 2005Co-Authors: B E Wiggins, Linda L KinkelAbstract:ABSTRACT A 2-year field trial was conducted to determine the effects of green manures and crop sequences on potato scab and Verticillium wilt. In addition, indigenous streptomycete densities and in vitro pathogen inhibitory activity were measured and their relationships to plant disease were determined. Green manures (buckwheat, canola, or fallow controls) were tested in conjunction with three crop sequences (alfalfa-potato, cornpotato, and potato-potato). Compared with fallow controls, tubers grown in buckwheat-treated soil had significantly lower Verticillium wilt ratings, and tubers grown in buckwheat- or canola-treated soil had greater yields. Potatoes grown in soil planted to corn or alfalfa the previous year had significantly lower Verticillium wilt and potato scab ratings as well as higher yields than potatoes grown in soil previously planted to potato. Streptomycetes from soils collected from green manure-treated plots tended to have greater in vitro pathogen inhibitory activity than streptomycete...
