scholarly journals Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

2005 ◽  
Vol 71 (5) ◽  
pp. 2452-2459 ◽  
Author(s):  
Alessandra S. Eustáquio ◽  
Bertolt Gust ◽  
Ute Galm ◽  
Shu-Ming Li ◽  
Keith F. Chater ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Attachment Site ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Gene Deletions ◽  
Or Gene ◽  
Genetically Manipulated

ABSTRACT A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

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 Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 424
Author(s):  
Osama G. Mohamed ◽  
Sadaf Dorandish ◽  
Rebecca Lindow ◽  
Megan Steltz ◽  
Ifrah Shoukat ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Gene Clusters ◽  
Multidrug Resistant ◽  
Microbial Interactions ◽  
Mass Spectrometry Data ◽  
Metagenomic Data ◽  
Resistant Bacteria ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Plate Technique

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny Earth®, we isolated over 1400 antibiotic-producing microbes, including 62, showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

scholarly journals Phytoplankton trigger the production of cryptic metabolites in the marine actinobacteria Salinispora tropica

2020 ◽  
Author(s):  
Audam Chhun ◽  
Despoina Sousoni ◽  
Maria del Mar Aguiló-Ferretjans ◽  
Lijiang Song ◽  
Christophe Corre ◽  
...  
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Antibiotic Production ◽  
Antimicrobial Effect ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Marine Actinobacteria ◽  
Salinispora Tropica ◽  
Eukaryotic Phytoplankton

AbstractBacteria from the Actinomycete family are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacteria Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide A but half of its putative BGCs are still orphan. Although previous studies have looked into using marine heterotrophs to induce orphan BGCs in Salinispora, the potential impact of co-culturing marine phototrophs with Salinispora has yet to be investigated. Following the observation of clear antimicrobial phenotype of the actinobacterium on a range of phytoplanktonic organisms, we here report the discovery of novel cryptic secondary metabolites produced by S. tropica in response to its co-culture with photosynthetic primary producers. An approach combining metabolomics and proteomics revealed that the photosynthate released by phytoplankton influences the biosynthetic capacities of S. tropica with both production of new molecules and the activation of orphan BGCs. Our work pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from actinobacteria.ImportanceThe alarming increase of antimicrobial resistance has generated an enormous interest in the discovery of novel active compounds. The isolation of new microbes to untap novel natural products is currently hampered because most biosynthetic gene clusters (BGC) encoded by these microorganisms are not expressed under standard laboratory conditions, i.e. mono-cultures. Here we show that co-culturing can be an easy way for triggering silent BGC. By combining state-of-the-art metabolomics and high-throughput proteomics, we characterized the activation of cryptic metabolites and silent biosynthetic gene clusters in the marine actinobacteria Salinispora tropica by the presence of phytoplankton photosynthate. We further suggest a mechanistic understanding of the antimicrobial effect this actinobacterium has on a broad range of prokaryotic and eukaryotic phytoplankton species and reveal a promising candidate for antibiotic production.

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 Competition sensing alters antibiotic production in Streptomyces

2020 ◽  
Author(s):  
Sanne Westhoff ◽  
Alexander Kloosterman ◽  
Stephan F. A. van Hoesel ◽  
Gilles P. van Wezel ◽  
Daniel E. Rozen
Keyword(s):  
Genetic Relatedness ◽  
Cell Damage ◽  
Antibiotic Production ◽  
Interference Competition ◽  
Resource Limitation ◽  
Gene Clusters ◽  
Regulatory Control ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
The Social

ABSTRACTOne of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require “elicitors” to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing streptomycetes we show that inhibition between competitors is common and occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreased in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competion. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes.SIGNIFICANCE STATEMENTBacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in Streptomyces, bacteria renowned for their production of antibiotics. We show that Streptomyces are more likely to produce antibiotics when grown with closely related competitors or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor’s toxins, in contrast to predictions of the competition sensing hypothesis. Streptomyces also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.

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