scholarly journals Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

2016 ◽  
Vol 82 (19) ◽  
pp. 5795-5805 ◽  
Author(s):  
Min Xu ◽  
Yemin Wang ◽  
Zhilong Zhao ◽  
Guixi Gao ◽  
Sheng-Xiong Huang ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Functional Screening ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type ◽  
Bac Libraries

ABSTRACTGenome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries inStreptomycesspp. We demonstrate mining from a strain ofStreptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate hostStreptomyces lividansSBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic fromS. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways.IMPORTANCEMicrobial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites fromStreptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic fromStreptomyces rocheiSal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery.

scholarly journals Recent advances in the genome mining of Aspergillus secondary metabolites (covering 2012–2018)

MedChemComm ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 840-866 ◽  
Author(s):  
Jillian Romsdahl ◽  
Clay C. C. Wang
Keyword(s):  
Aspergillus Terreus ◽  
Genome Mining ◽  
Gene Clusters ◽  
Genetic Identification ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
The Past ◽  
Made In

This review covers advances made in genome mining SMs produced by Aspergillus nidulans, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus in the past six years (2012–2018). Genetic identification and molecular characterization of SM biosynthetic gene clusters, along with proposed biosynthetic pathways, is discussed in depth.

scholarly journals Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

2010 ◽  
Vol 76 (8) ◽  
pp. 2681-2683 ◽  
Author(s):  
D. Cole Stevens ◽  
Michael R. Henry ◽  
Kimberly A. Murphy ◽  
Christopher N. Boddy
Keyword(s):  
Drug Discovery ◽  
Heterologous Expression ◽  
Biosynthetic Pathway ◽  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Metagenomic Dna ◽  
Dna Libraries

ABSTRACT New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.

scholarly journals Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059

2021 ◽  
Author(s):  
Xiyan Wang ◽  
Thomas Isbrandt ◽  
Emil Ørsted Christensen ◽  
Jette Melchiorsen ◽  
Thomas Ostenfeld Larsen ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Pseudoalteromonas Rubra

Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression.

scholarly journals Genome Mining and Characterization of Biosynthetic Gene Clusters in Two Cave Strains of Paenibacillus sp.

2021 ◽  
Vol 11 ◽  
Author(s):  
Jolanta Lebedeva ◽  
Gabriele Jukneviciute ◽  
Rimvydė Čepaitė ◽  
Vida Vickackaite ◽  
Raminta Pranckutė ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Phenotypic Characterization ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Paenibacillus Sp ◽  
Peptide Synthetase ◽  
Non Ribosomal Peptides

The genome sequencing and mining of microorganisms from unexplored and extreme environments has become important in the process of identifying novel biosynthetic pathways. In the present study, the biosynthetic potential of Paenibacillus sp. strains 23TSA30-6 and 28ISP30-2 was investigated. Both strains were isolated from the deep oligotrophic Krubera-Voronja Cave and were found to be highly active against both Gram-positive and Gram-negative bacteria. Genome mining revealed a high number of biosynthetic gene clusters in the cave strains: 21 for strain 23TSA30-6 and 19 for strain 28ISP30-2. Single clusters encoding the biosynthesis of phosphonate, terpene, and siderophore, as well as a single trans-AT polyketide synthase/non-ribosomal peptide synthetase, were identified in both genomes. The most numerous clusters were assigned to the biosynthetic pathways of non-ribosomal peptides and ribosomally synthesized and post-translationally modified peptides. Although four non-ribosomal peptide synthetase gene clusters were predicted to be involved in the biosynthesis of known compounds (fusaricidin, polymyxin B, colistin A, and tridecaptin) of the genus Paenibacillus, discrepancies in the structural organization of the clusters, as well as in the substrate specificity of some adenylation domains, were detected between the reference pathways and the clusters in our study. Among the clusters involved in the biosynthesis of ribosomally synthesized peptides, only one was predicted to be involved in the biosynthesis of a known compound: paenicidin B. Most biosynthetic gene clusters in the genomes of the cave strains showed a low similarity with the reference pathways and were predicted to represent novel biosynthetic pathways. In addition, the cave strains differed in their potential to encode the biosynthesis of a few unique, previously unknown compounds (class II lanthipeptides and three non-ribosomal peptides). The phenotypic characterization of proteinaceous and volatile compounds produced by strains 23TSA30-6 and 28ISP30-2 was also performed, and the results were compared with those of genome mining.

scholarly journals Genome Mining Uncovers Clustered Biosynthetic Pathways for Defense-Related Molecules in Bread Wheat

2021 ◽  
Author(s):  
Guy Polturak ◽  
Martin Dippe ◽  
Michael J Stephenson ◽  
Rajesh Chandra Misra ◽  
Charlotte Owen ◽  
...  
Keyword(s):  
Stress Responses ◽  
Regulatory Network ◽  
Genome Mining ◽  
Gene Clusters ◽  
Chemical Defenses ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Yield Losses ◽  
Biosynthetic Gene Clusters ◽  
The World

Wheat is one of the most widely grown food crops in the world. However, it succumbs to numerous pests and pathogens that cause substantial yield losses. A better understanding of biotic stress responses in wheat is thus of major importance. Here we identify previously unknown pathogen-induced biosynthetic pathways that produce a diverse set of molecules, including flavonoids, diterpenes and triterpenes. These pathways are encoded by six biosynthetic gene clusters and share a common regulatory network. We further identify associations with known or novel phytoalexin clusters in other cereals and grasses. Our results significantly advance the understanding of chemical defenses in wheat and open up new avenues for enhancing disease resistance in this agriculturally important crop.

scholarly journals Characterization and Engineering of Streptomyces griseofuscus DSM 40191 as a Potential Host for Heterologous Expression of Biosynthetic Gene Clusters

2020 ◽  
Author(s):  
Tetiana Gren ◽  
Christopher M. Whitford ◽  
Omkar S. Mohite ◽  
Tue S. Jørgensen ◽  
Eftychia E. Kontou ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Phosphorus Sources ◽  
Further Development ◽  
Core Genes ◽  
Target Effects

AbstractStreptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Using CRISPR-BEST technology, core genes of 4 biosynthetic gene clusters (BGCs) that are situated on the chromosome arms were inactivated and the outcomes of the inactivations were tested. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5,19% genome reduction. Sequencing confirmed that DEL2 does not bear any crucial off-target effects or rearrangements in its genome. It can be characterized by faster growth and inability to produce three main native metabolites of S. griseofuscus: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to confirm the production of actinorhodin by both S. griseofuscus wild type and DEL2. We believe that this strain will serve as a good chassis for heterologous expression of BGCs.ImportanceThe rise of antibacterial resistance calls on the development of the next generation of antibiotics, majority of which are derived from natural compounds, produced by actinomycetes. The manipulation, refactoring and expression of BGCs coding for such natural products is a promising approach in secondary metabolite discovery. Thus, the development of a versatile panel of heterologous hosts for the expression of BGCs is essential. We believe that first-to-date systematic, detailed characterisation of S. griseofuscus, a highly promising chassis strain, will not only facilitate the further development of this particular strain, but also will set a blueprint for characterisation of other potential hosts.

scholarly journals Coordinate Regulation of Antimycin and Candicidin Biosynthesis

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Thomas C. McLean ◽  
Paul A. Hoskisson ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Secondary Metabolism ◽  
Gene Clusters ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type ◽  
Regulatory Strategies ◽  
Pathway Activation ◽  
Drug Leads

ABSTRACT Natural products produced by members of the phylum Actinobacteria underpin many industrially and medically important compounds; however, the majority of the ~30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. Understanding the diversity of regulatory strategies controlling the expression of these pathways is therefore critical if their biosynthetic potential is to be explored for new drug leads. Our findings reveal that the candicidin cluster-situated regulator FscRI coordinately controls the biosynthesis of both candicidin and antimycin, which is the first observation of cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products. We anticipate that this will emerge as a major strategy by which members of the phylum Actinobacteria coordinately produce natural products, which will advance our understanding of how the expression of secondary metabolism is controlled and will aid the pursuit of “silent” biosynthetic pathway activation. Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a few them are expressed in the laboratory; thus, poor understanding of how natural-product biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species, and their regulation is atypical compared to that of most other natural products. Here we demonstrate that antimycin production by Streptomyces albus S4 is regulated by FscRI, a PAS-LuxR family cluster-situated regulator of the polyene antifungal agent candicidin. We report that heterologous production of antimycins by Streptomyces coelicolor is dependent on FscRI and show that FscRI activates the transcription of key biosynthetic genes. We also demonstrate through chromatin immunoprecipitation sequencing that FscRI regulation is direct, and we provide evidence that this regulation strategy is conserved and unique to short-form antimycin gene clusters. Our study provides direct in vivo evidence of the cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products and expands the paradigmatic understanding of the regulation of secondary metabolism. IMPORTANCE Natural products produced by members of the phylum Actinobacteria underpin many industrially and medically important compounds; however, the majority of the ~30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. Understanding the diversity of regulatory strategies controlling the expression of these pathways is therefore critical if their biosynthetic potential is to be explored for new drug leads. Our findings reveal that the candicidin cluster-situated regulator FscRI coordinately controls the biosynthesis of both candicidin and antimycin, which is the first observation of cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products. We anticipate that this will emerge as a major strategy by which members of the phylum Actinobacteria coordinately produce natural products, which will advance our understanding of how the expression of secondary metabolism is controlled and will aid the pursuit of “silent” biosynthetic pathway activation.

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