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Synthetic and Systems Biotechnology Sep 2022Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems. J1074 is a model organism used in fundamental...
Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems. J1074 is a model organism used in fundamental research and industrial microbiology. Nevertheless, a comprehensive and standardized method for analyzing the metabolome of is yet to be developed. Thus, we comprehensively evaluated and optimized the analytical procedure and sample preparation for profiling polar metabolites using hydrophilic interaction liquid chromatography (HILIC) coupled with high-resolution mass spectrometry (HRMS). We systematically examined the HILIC columns, quenching solutions, sample-to-quenching ratios, and extraction methods. Then, the optimal protocol was used to investigate the dynamic intracellular polar metabolite profile of the engineered J1074 strains during spinosad (spinosyn A and spinosyn D) fermentation. A total of 3648 compounds were detected, and 83 metabolites were matched to the standards. The intracellular metabolomic profiles of engineered J1074 strains (ADE-AP and OE3) were detected; furthermore, their metabolomes in different stages were analyzed to reveal the reasons for their differences in their spinosad production, as well as the current metabolic limitation of heterologous spinosad production in J1074. The HILIC-HRMS method is a valuable tool for investigating polar metabolomes, and provides a reference methodology to study other metabolomes
PubMed: 35664928
DOI: 10.1016/j.synbio.2022.05.004 -
The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4.Archives of Microbiology Apr 2022In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of...
In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270-4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511-520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).
Topics: Animals; Antifungal Agents; Ants; Candicidin; Polyenes; Streptomyces
PubMed: 35488016
DOI: 10.1007/s00203-022-02906-w -
Antibiotics (Basel, Switzerland) Mar 2018Rishirilide B was isolated from and on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by...
Rishirilide B was isolated from and on the basis of its inhibitory activity towards alpha-2-macroglobulin. The biosynthesis of rishirilide B was investigated by feeding experiments with different C labelled precursors using the heterologous host J1074::cos4 containing a cosmid encoding of the gene cluster responsible for rishirilide B production. NMR spectroscopic analysis of labelled compounds demonstrate that the tricyclic backbone of rishirilide B is a polyketide synthesized from nine acetate units. One of the acetate units is decarboxylated to give a methyl group. The origin of the starter unit was determined to be isobutyrate.
PubMed: 29518990
DOI: 10.3390/antibiotics7010020 -
Advances in Clinical and Experimental... Jul 2018According to numerous studies, failures in treatment of ovarian cancer, i.e., a relapse and metastases, result from a small population of cancer stem cells (CSCs). They... (Review)
Review
According to numerous studies, failures in treatment of ovarian cancer, i.e., a relapse and metastases, result from a small population of cancer stem cells (CSCs). They may also be responsible for tumor initiation. Cancer stem cells are resistant to chemoand radiotherapy. Eradication of CSCs may involve the application of salinomycin, metformin and Clostridium perfringens; the effect of anti-angiogenic factors remains controversial. Salinomycin is an antibiotic isolated from Streptomyces albus bacteria. Its CSC-eradicating effect has been demonstrated both in ovarian cancer cell lines and in women with breast cancer. Clostridium perfringens enterotoxin (CPE) has been demonstrated to destroy CSCs in ovarian cancer both in vivo and in vitro. Metformin, apart from its hypoglycemic effect, reduces the CSC population and inhibits the proliferation of neoplastic cells and angiogenesis. Cancer stem cells with expression of VEGFR1+ have been described as affecting circulating cancer cells and influencing the formation of metastases. Both positive and negative effects of anti-angiogenic therapy on the CSC population have been documented.
Topics: Animals; Cell Proliferation; Enterotoxins; Female; Humans; Metformin; Neoplastic Stem Cells; Ovarian Neoplasms; Pyrans
PubMed: 29938937
DOI: 10.17219/acem/73999 -
Molecules (Basel, Switzerland) Oct 2021Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High...
Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.
Topics: Bacterial Proteins; Biocatalysis; Drug Discovery; Flavonoids; Frankia; Genes, Bacterial; Halogenation; Molecular Docking Simulation; Oxidoreductases; Recombinant Proteins; Substrate Specificity
PubMed: 34684801
DOI: 10.3390/molecules26206220 -
Cytology and Genetics 2022The J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from...
The J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the gene encoding a ribosomal protein S12. The interest in such mutations in actinobacteria is due to the fact that they provide a considerable increase in the antibiotic activity. In this work, we describe the isolation and characterization of the KO-1297 strain, which contains a spontaneous missense mutation in the gene leading to a Lys88Glu substitution in the protein S12. As compared with the initial strain, this mutant exhibits an increased resistance to streptomycin and higher antibiotic productivity. The KO-1297 strain and genetically engineered mutant K88E are not identical in their ability to produce antibiotics. KO-1297 also exhibits a certain level of instability of mutation. The genomes of KO-1297 and its revertant contain the mutations that can cause phenotypic differences between these strains (as well as between them and SAM2 and K88E strains).
PubMed: 35194265
DOI: 10.3103/S009545272201011X -
Synthetic Biology (Oxford, England) 2022In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of...
In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5 regulatory sequences (ARES) consisting of promoters and 5 untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, and , and the eukaryote yeast .
PubMed: 36212995
DOI: 10.1093/synbio/ysac017 -
Scientific Reports Feb 2023In this study, we identified a suitable precursor and good cellular compartmentalization for enhancing bioactive metabolites to produce biosynthetic zinc oxide...
Scaling-up strategies for controllable biosynthetic ZnO NPs using cell free-extract of endophytic Streptomyces albus: characterization, statistical optimization, and biomedical activities evaluation.
In this study, we identified a suitable precursor and good cellular compartmentalization for enhancing bioactive metabolites to produce biosynthetic zinc oxide nanoparticles (ZnO NPs). An effective medium for cultivating endophytic Streptomyces albus strain E56 was selected using several optimized approaches in order to maximize the yield of biosynthetic ZnO NPs. The highest biosynthetic ZnO NPs yield (4.63 g/L) was obtained when pipetting the mixed cell-free fractions with 100 mM of zinc sulfate as a precursor. The generation of biosynthetic ZnO NPs was quickly verified using a colored solution (white color) and UV-Visible spectroscopy (maximum peak, at 320 nm). On a small scale, the Taguchi method was applied to improve the culture medium for culturing the strain E56. As a result, its cell-dry weight was 3.85 times that of the control condition. And then the biosynthesis of ZnO NPs (7.59 g/L) was increased by 1.6 times. Furthermore, by using the Plackett-Burman design to improve the utilized biogenesis pathway, the biosynthesis of ZnO NPs (18.76 g/L) was increased by 4.3 times. To find the best growth production line, we used batch and fed batch fermentation modes to gradually scale up biomass output. All kinetics of studied cell growth were evaluated during fed-batch fermentation as follows: biomass yield was 271.45 g/L, yield coefficient was 94.25 g/g, and ZnO NPs yield was 345.32 g/L. In vitro, the effects of various dosages of the controllable biosynthetic ZnO NPs as antimicrobial and anticancer agents were also investigated. The treatments with controllable biosynthetic ZnO NPs had a significant impact on all the examined multidrug-resistant human pathogens as well as cancer cells.
Topics: Humans; Zinc Oxide; Metal Nanoparticles; Anti-Infective Agents; Plant Extracts
PubMed: 36823304
DOI: 10.1038/s41598-023-29757-9 -
Applied and Environmental Microbiology May 2018In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as hormones based on their extremely low...
In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that strain J1074 may produce butenolide-type hormones. Here, we describe metabolite profiling of a disruptant of the gene, which encodes a key biosynthetic enzyme for butenolide-type hormones, and identify four butenolide compounds from J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for metabolic profiling demonstrated that the butenolide compounds of J1074 stimulate avermectin production in another species, , illustrating the complex chemical interactions through interspecies signals in streptomycetes. Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type hormones. Moreover, the diffusible butenolide compounds from a donor strain were recognized by the recipient strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.
Topics: 4-Butyrolactone; Bacterial Proteins; Gene Expression; Ivermectin; Mass Spectrometry; Metabolome; Secondary Metabolism; Species Specificity; Streptomyces
PubMed: 29500256
DOI: 10.1128/AEM.02791-17 -
Frontiers in Oncology 2021Breast cancer (BC) is the most frequent cancer among women worldwide and is the leading cause of cancer-related deaths in women. Cancer cells with stem cell-like... (Review)
Review
Breast cancer (BC) is the most frequent cancer among women worldwide and is the leading cause of cancer-related deaths in women. Cancer cells with stem cell-like features and tumor-initiating potential contribute to drug resistance, tumor recurrence, and metastasis. To achieve better clinical outcomes, it is crucial to eradicate both bulk BC cells and breast cancer stem cells (BCSCs). Salinomycin, a monocarboxylic polyether antibiotic isolated from , can precisely kill cancer stem cells (CSCs), particularly BCSCs, by various mechanisms, including apoptosis, autophagy, and necrosis. There is increasing evidence that salinomycin can inhibit cell proliferation, invasion, and migration in BC and reverse the immune-inhibitory microenvironment to prevent tumor growth and metastasis. Therefore, salinomycin is a promising therapeutic drug for BC. In this review, we summarize established mechanisms by which salinomycin protects against BC and discuss its future clinical applications.
PubMed: 34381705
DOI: 10.3389/fonc.2021.654428