scholarly journals Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping Men ◽  
Min Wang ◽  
Jinda Li ◽  
Ce Geng ◽  
Xuenian Huang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Secondary Metabolites ◽  
Polyketide Synthase ◽  
Genetic Manipulation ◽  
Fungal Infections ◽  
Genome Mining ◽  
Transformation Method ◽  
Selectable Markers ◽  
Cyclic Hexapeptide ◽  
Manipulation System

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

scholarly journals Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the HaloarchaeonNatrinemasp. J7-2

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Jie Lv ◽  
Shuai Wang ◽  
Yuchen Wang ◽  
Yuping Huang ◽  
Xiangdong Chen
Keyword(s):  
Genomic Dna ◽  
Genetic Manipulation ◽  
Gene Mutations ◽  
Transformation Method ◽  
Wild Type ◽  
Auxotrophic Mutants ◽  
Archaeal Species ◽  
Manipulation System ◽  
Exogenous Genes ◽  
Mutant Phenotypes

Our understanding of the genusNatrinemais presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants ofNatrinemasp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations inleuB(J7-2-1),dapD(J7-2-26), andargC(J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103transformants perµg DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes,bgaHoramyHgene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for theNatrinemagenus based on the isolated auxotrophic mutants ofNatrinemasp. J7-2.

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

scholarly journals Protective Antifungal Activity of Plantago major Extract Against the Phytopathogenic Fungi Phytophthora cinnamomi, Diplodia corticola and Colletotrichum Species

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 94
Author(s):  
Cláudia Ferreira ◽  
Rui Oliveira
Keyword(s):  
Antifungal Activity ◽  
Secondary Metabolites ◽  
Plant Extracts ◽  
Phytophthora Cinnamomi ◽  
Fungal Infections ◽  
Petri Dish ◽  
Phytopathogenic Fungi ◽  
Colletotrichum Acutatum ◽  
Lower Probability ◽  
Plantago Major

Synthetic fungicides for crops protection raise environmental and human concerns due to accumulation in edible vegetables, showing significant toxicity to humans, and in soil, groundwater and rivers, affecting ecological balance. In addition, they are prone to the development of resistant strains because of the single target-based mechanism of action. Plant extracts provide attractive alternatives, as they constitute a rich source of biodegradable secondary metabolites, such as phenols, flavonoids and saponins, which have multiple modes of antifungal action and a lower probability of the development of resistant fungi. This work has the objective of identifying plant extracts with antifungal activity, aiming to contribute to food safety and sustainable agricultural practices. We selected a saponin-containing plant, Plantago major, and extracted secondary metabolites with 50% (v/v) ethanol, dried by evaporation, and dissolved in water. For antifungal activity, the phytopathogenic fungi Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum nymphaeae, Diplodia corticola and Phytophthora cinnamomi were selected because they affect fruits and vegetables, such as strawberry, almond, apple, avocado, blueberry and chestnut trees. The aqueous extract was incorporated into PDA medium at different concentrations and mycelial discs were placed in the center of each Petri dish. Growth was measured as the radial mycelial growth at 3, 6, and 9 days incubation at 25 °C in the dark. The maximum growth inhibition (32.2%) was obtained against P. cinnamomi with 2000 µg/mL extract followed by C. gloeosporioides (25.7%) on the sixth day and by C. godetiae and C. nymphaeae (21.1%) on the ninth day. Results show that P. major presents antifungal activity in all phytopathogenic fungi tested and the extract can be used to protect important crops, by inhibiting the development of fungal infections and promoting food security and a sustainable agriculture.

scholarly journals Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 108
Author(s):  
Xiyan Wang ◽  
Thomas Isbrandt ◽  
Mikael Lenz Strube ◽  
Sara Skøtt Paulsen ◽  
Maike Wennekers Nielsen ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Genetic Manipulation ◽  
Genome Mining ◽  
Hydrolytic Enzymes ◽  
Metabolite Profile ◽  
Chitinolytic Enzyme ◽  
Chitin Degradation ◽  
Metabolite Profiles ◽  
Bioactive Potential ◽  
Pseudoalteromonas Rubra

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

scholarly journals Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas

2015 ◽  
Vol 14 (1) ◽  
pp. 11 ◽  
Author(s):  
Pengxia Wang ◽  
Zichao Yu ◽  
Baiyuan Li ◽  
Xingsheng Cai ◽  
Zhenshun Zeng ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Manipulation System

scholarly journals Genome mining of ant-associated bacteria to identify novel secondary metabolites

2019 ◽  
Vol 1 (1A) ◽  
Author(s):  
Jacob Hamilton ◽  
Neil Holmes ◽  
Sarah Worsley ◽  
Elaine Patrick ◽  
Naomi Pierce ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genome Mining ◽  
Associated Bacteria

scholarly journals Antimicrobial compounds were isolated from the secondary metabolites of endophytic Gordonia in the intestinal tract of Periplaneta americana

2021 ◽  
Author(s):  
Yan Ma ◽  
Minhua Xu ◽  
Hancong Liu ◽  
Tiantian Yu ◽  
Ping Guo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Secondary Metabolites ◽  
Intestinal Tract ◽  
Periplaneta Americana ◽  
Actinomycin D ◽  
Fungal Infections ◽  
Early Stage ◽  
Phylogenetic Analyses ◽  
Morphological Characteristics ◽  
Antimicrobial Compounds

Abstract Background: As we all know, bacterial and fungal infections have become one of the threats to human health. Microbial secondary metabolites are one of the main sources of bioactive natural products. It is estimated that around 60% of all foregone antibiotics are derived from secondary metabolites produced by filamentous actinomycete bacteria. Gordonia spp. are members of the actinomycete family, their contribution to the environment improvement and environmental protection by their biological degradation ability, but there are few studies on their antimicrobial activity of their secondary metabolites. Our team isolated a Gordonia strain WA 4-31 with anti-Candida albicans activity from the intestinal tract of Periplaneta americana in the early stage.Results: In this study, we firstly identified the strain WA 4-31 by the morphological characteristics and the phylogenetic analyses, and found that it homologous to a strain of Gordonia from the Indian desert (EU333873) by 100%. Then four compounds, Actinomycin D (1), Actinomycin X2 (2), Mojavensin A (3) and cyclic (leucine-leucne) dipeptide (4) were purified from the EtOH extract of the fermented broth of the strain. The compounds 1-4 had activities against Candida albicans, Aspergillus niger, Aspergillus fumigatus and Trichophyton rubrum. They also had activities against MRSA, S.aureus, K.peneumoniae and E.coli in different degree. The minimum inhibitory concentration of Actinomycin D and Actinomycin X2 on MASA was 0.25 μg/mL. Interestingly, we found that when Mojavensin A was mixed with compound 4 ratio of 1:1, the solution of the compounds was better than the single on anti-Candida albicans. Besides, compounds 1-3 had varying degrees of cytotoxicity on CNE-2 cells and HepG-2 cells.Conclusions: The present study firstly reported the antimicrobial compounds isolated from Gordonia. These indicated that rare actinomycetes from the intestinal tract of Periplaneta americana possessed a potential as a source of active secondary metabolites.

Synthesis of the C50 Diastereomers of the C33-C51 Fragment of Stambomycin D

2022 ◽  
Author(s):  
Yongchen Wang ◽  
Venkaiah Chintalapudi ◽  
Haraldur Gudmundsson ◽  
Gregory L Challis ◽  
Edward Alexander Anderson
Keyword(s):  
Sequence Analysis ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Macrolide Antibiotics ◽  
Stereochemical Assignment

As products of genome mining, the stereochemical assignment of the macrolide antibiotics stambomycins A–D has been made on the basis of sequence analysis of the associated polyketide synthase, aside from...

scholarly journals Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

2020 ◽  
Vol 11 ◽  
Author(s):  
Jin Lü ◽  
Qingshan Long ◽  
Zhilong Zhao ◽  
Lu Chen ◽  
Weijun He ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Bac Library ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Saccharopolyspora Erythraea ◽  
Heterologous Production ◽  
Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

scholarly journals Genomic and Metabolomic Analysis of Antarctic Bacteria Revealed Culture and Elicitation Conditions for the Production of Antimicrobial Compounds

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 673
Author(s):  
Kattia Núñez-Montero ◽  
Damián Quezada-Solís ◽  
Zeinab G. Khalil ◽  
Robert J. Capon ◽  
Fernando D. Andreote ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Secondary Metabolites ◽  
Culture Media ◽  
Genome Mining ◽  
Gene Clusters ◽  
Culture Conditions ◽  
Bioactive Metabolites ◽  
Bacterial Strains ◽  
Antarctic Bacteria ◽  
New Antibiotics

Concern about finding new antibiotics against drug-resistant pathogens is increasing every year. Antarctic bacteria have been proposed as an unexplored source of bioactive metabolites; however, most biosynthetic gene clusters (BGCs) producing secondary metabolites remain silent under common culture conditions. Our work aimed to characterize elicitation conditions for the production of antibacterial secondary metabolites from 34 Antarctic bacterial strains based on MS/MS metabolomics and genome mining approaches. Bacterial strains were cultivated under different nutrient and elicitation conditions, including the addition of lipopolysaccharide (LPS), sodium nitroprusside (SNP), and coculture. Metabolomes were obtained by HPLC-QTOF-MS/MS and analyzed through molecular networking. Antibacterial activity was determined, and seven strains were selected for genome sequencing and analysis. Biosynthesis pathways were activated by all the elicitation treatments, which varies among strains and dependents of culture media. Increased antibacterial activity was observed for a few strains and addition of LPS was related with inhibition of Gram-negative pathogens. Antibiotic BGCs were found for all selected strains and the expressions of putative actinomycin, carotenoids, and bacillibactin were characterized by comparison of genomic and metabolomic data. This work established the use of promising new elicitors for bioprospection of Antarctic bacteria and highlights the importance of new “-omics” comparative approaches for drug discovery.

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