Biosynthetic strategies for tetramic acid formation

Serratia strains are ubiquitous microorganisms with the ability to produce serratomolides, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi and may have a nematicidal activity against phytopathogenic nematodes. Serrawettins W1 and W2 from different strains have different structures that might be correlated with distinct genomic organizations. This work used comparative genomics to determine the distribution and the organization of the serrawettins biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. The serrawettin W1 and W2 gene clusters’ organization was established using antiSMASH software and compared with single and short data previously described for YD25TSerratia. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW was present in 17 Serratia genomes. Eighty different coding sequence (CDS) were assigned to the W1 gene cluster, 13 being common to all clusters. The serrawettin W2 swrA gene was present in 11 Serratia genomes. The W2 gene clusters included 68 CDS with 24 present in all the clusters. The genomic analysis showed the swrA gene constitutes five modules, four with three domains and one with four domains, while the swrW gene constitutes one module with four domains. This work identified four genes common to all serrawettin gene clusters, highlighting their essential potential in the serrawettins biosynthetic process.

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A Regulator Based “Semi-Targeted” Approach to Activate Silent Biosynthetic Gene Clusters

International Journal of Molecular Sciences ◽

10.3390/ijms22147567 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7567

Author(s):

Erik Mingyar ◽

Lucas Mühling ◽

Andreas Kulik ◽

Anika Winkler ◽

Daniel Wibberg ◽

...

Keyword(s):

Gene Clusters ◽

Regulatory Proteins ◽

Biosynthetic Gene ◽

Standard Laboratory ◽

Biosynthetic Gene Clusters ◽

Streptomyces Sp ◽

Strain Collection ◽

Regulator Genes ◽

The Individual ◽

Targeted Approach

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

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Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Patrick Videau ◽

Kaitlyn Wells ◽

Arun Singh ◽

Jessie Eiting ◽

Philip Proteau ◽

...

Keyword(s):

Natural Products ◽

Genome Mining ◽

Gene Clusters ◽

Combinatorial Biosynthesis ◽

Test Case ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

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Faculty Opinions recommendation of A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718872945.793500860 ◽

2014 ◽

Author(s):

Howard Young ◽

Heekyong Bae

Keyword(s):

Human Microbiome ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Systematic Analysis

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Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-019-02198-y ◽

2019 ◽

Vol 46 (9-10) ◽

pp. 1381-1400 ◽

Cited By ~ 10

Author(s):

Hailey A. Tomm ◽

Lorena Ucciferri ◽

Avena C. Ross

Keyword(s):

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Metabolite Production ◽

Culturing Conditions

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Global biogeographic sampling of bacterial secondary metabolism

eLife ◽

10.7554/elife.05048 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 74

Author(s):

Zachary Charlop-Powers ◽

Jeremy G Owen ◽

Boojala Vijay B Reddy ◽

Melinda A Ternei ◽

Denise O Guimarães ◽

...

Keyword(s):

Secondary Metabolites ◽

Genome Sequencing ◽

Geographic Distance ◽

Gene Clusters ◽

Natural World ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Road Map ◽

Two Factors ◽

Natural Products Discovery

Recent bacterial (meta)genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment. Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ketosynthase domains to compare biosynthetic diversity in soil microbiomes from around the globe. We see large differences in domain populations from all except the most proximal and biome-similar samples, suggesting that most microbiomes will encode largely distinct collections of bacterial secondary metabolites. Our data indicate a correlation between two factors, geographic distance and biome-type, and the biosynthetic diversity found in soil environments. By assigning reads to known gene clusters we identify hotspots of biomedically relevant biosynthetic diversity. These observations not only provide new insights into the natural world, they also provide a road map for guiding future natural products discovery efforts.

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Challenges of functional expression of complex polyketide biosynthetic gene clusters

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2020.12.007 ◽

2021 ◽

Vol 69 ◽

pp. 103-111

Author(s):

Yaojie Gao ◽

Yuchun Zhao ◽

Xinyi He ◽

Zixin Deng ◽

Ming Jiang

Keyword(s):

Gene Clusters ◽

Functional Expression ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters

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

Marine Drugs ◽

10.3390/md19080424 ◽

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.

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CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

Scientific Reports ◽

10.1038/s41598-020-80864-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

László Mózsik ◽

Mirthe Hoekzema ◽

Niels A. W. de Kok ◽

Roel A. L. Bovenberg ◽

Yvonne Nygård ◽

...

Keyword(s):

Filamentous Fungi ◽

Transcriptional Activation ◽

Core Promoter ◽

Crop Protection ◽

Gene Clusters ◽

Growth Conditions ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Fluorescent Reporter ◽

A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

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