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 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.

RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression

2016 ◽  
Vol 11 (7) ◽  
pp. 1175-1190 ◽  
Author(s):  
Hailong Wang ◽  
Zhen Li ◽  
Ruonan Jia ◽  
Yu Hou ◽  
Jia Yin ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Direct Cloning

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.

Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains

Biopolymers ◽  
2010 ◽  
Vol 93 (9) ◽  
pp. 823-832 ◽  
Author(s):  
Katrin Flinspach ◽  
Lucia Westrich ◽  
Leonard Kaysser ◽  
Stefanie Siebenberg ◽  
Juan Pablo Gomez-Escribano ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Genetically Modified ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

scholarly journals Understanding Thioamitide Biosynthesis Using Pathway Engineering and Untargeted Metabolomics

2020 ◽  
Author(s):  
Tom H. Eyles ◽  
Natalia M. Vior ◽  
Rodney Lacret ◽  
Andrew W. Truman
Keyword(s):  
Biosynthetic Pathway ◽  
Gene Clusters ◽  
Untargeted Metabolomics ◽  
Pathway Engineering ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Heterologous Host ◽  
Detailed Understanding ◽  
Precursor Peptide ◽  
Modified Peptides

ABSTRACTThiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the gene clusters of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 gene cluster in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified gene clusters were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway, including the identification of an effector-like protein critical for amino acid dehydration. We use this biosynthetic understanding to bioinformatically identify new widespread families of RiPP biosynthetic gene clusters, paving the way for future RiPP discovery and engineering.

scholarly journals Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

2018 ◽  
Author(s):  
Kwo-Kwang Abraham Wang ◽  
Tai L. Ng ◽  
Peng Wang ◽  
Zedu Huang ◽  
Emily P. Balskus ◽  
...  
Keyword(s):  
Natural Products ◽  
Nitrous Acid ◽  
Bond Formation ◽  
Gene Clusters ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Structural Differences ◽  
Related Compounds ◽  
Made In

AbstractFosfazinomycin and kinamycin are natural products that contain nitrogen-nitrogen (N-N) bonds but that are otherwise structurally unrelated. Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation. In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid. Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways. Therefore, unlike other pathways to NN bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products.

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