Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

In filamentous fungi, genes in secondary metabolite biosynthetic pathways are generally clustered. In the case of those pathways involved in nonribosomal peptide production, a nonribosomal peptide synthetase (NRPS) gene is commonly found as a main element of the cluster. Large multifunctional enzymes are encoded by members of this gene family that produce a broad spectrum of bioactive compounds. In this research, we applied genome-based identification of nonribosomal peptide biosynthetic gene clusters in the family Ceratocystidaceae. For this purpose, we used the whole genome sequences of species from the genera Ceratocystis, Davidsoniella, Thielaviopsis, Endoconidiophora, Bretziella, Huntiella, and Ambrosiella. To identify and characterize the clusters, different bioinformatics and phylogenetic approaches, as well as PCR-based methods were used. In all genomes studied, two highly conserved NRPS genes (one monomodular and one multimodular) were identified and their potential products were predicted to be siderophores. Expression analysis of two Huntiella species (H. moniliformis and H. omanensis) confirmed the accuracy of the annotations and proved that the genes in both clusters are expressed. Furthermore, a phylogenetic analysis showed that both NRPS genes of the Ceratocystidaceae formed distinct and well supported clades in their respective phylograms, where they grouped with other known NRPSs involved in siderophore production. Overall, these findings improve our understanding of the diversity and evolution of NRPS biosynthetic pathways in the family Ceratocystidaceae.

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Activation of a Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic Nonribosomal Peptide Synthetase Gene Cluster

Applied and Environmental Microbiology ◽

10.1128/aem.00683-10 ◽

2010 ◽

Vol 76 (24) ◽

pp. 8143-8149 ◽

Cited By ~ 96

Author(s):

Sebastian Bergmann ◽

Alexander N. Funk ◽

Kirstin Scherlach ◽

Volker Schroeckh ◽

Ekaterina Shelest ◽

...

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Secondary Metabolite ◽

Cross Talk ◽

Regulatory Gene ◽

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Nonribosomal Peptide ◽

Peptide Synthetase ◽

Biosynthesis Gene

ABSTRACT Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion of the asperfuranone activator gene afoA, indicating that ScpR binds to the afoA promoter. To the best of our knowledge, this is the first report of regulatory cross talk between two biosynthesis gene clusters located on different chromosomes.

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Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705016114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7025-7030 ◽

Cited By ~ 31

Author(s):

Nicholas C. Harris ◽

Michio Sato ◽

Nicolaus A. Herman ◽

Frederick Twigg ◽

Wenlong Cai ◽

...

Keyword(s):

Gene Cluster ◽

Acyl Chain ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptide Synthetase ◽

Biosynthetic Gene ◽

Nonribosomal Peptide ◽

Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

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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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Antimicrobials Inspired by Nonribosomal Peptide Synthetase Gene Clusters

Journal of the American Chemical Society ◽

10.1021/jacs.6b11861 ◽

2017 ◽

Vol 139 (4) ◽

pp. 1404-1407 ◽

Cited By ~ 18

Author(s):

Xavier Vila-Farres ◽

John Chu ◽

Daigo Inoyama ◽

Melinda A. Ternei ◽

Christophe Lemetre ◽

...

Keyword(s):

Gene Clusters ◽

Nonribosomal Peptide Synthetase ◽

Nonribosomal Peptide ◽

Peptide Synthetase

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Comprehensive curation and analysis of fungal biosynthetic gene clusters of published natural products

Fungal Genetics and Biology ◽

10.1016/j.fgb.2016.01.012 ◽

2016 ◽

Vol 89 ◽

pp. 18-28 ◽

Cited By ~ 64

Author(s):

Yong Fuga Li ◽

Kathleen J.S. Tsai ◽

Colin J.B. Harvey ◽

James Jian Li ◽

Beatrice E. Ary ◽

...

Keyword(s):

Natural Products ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters

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

10.1101/2020.05.18.103358 ◽

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.

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New Approaches to Detect Biosynthetic Gene Clusters in the Environment

Medicines ◽

10.3390/medicines6010032 ◽

2019 ◽

Vol 6 (1) ◽

pp. 32 ◽

Cited By ~ 7

Author(s):

Ray Chen ◽

Hon Wong ◽

Brendan Burns

Keyword(s):

Natural Products ◽

Secondary Metabolites ◽

Gene Clusters ◽

Screening Methods ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Diverse Range ◽

Bioinformatic Tools ◽

Culture Independent ◽

Traditional Approaches

Microorganisms in the environment can produce a diverse range of secondary metabolites (SM), which are also known as natural products. Bioactive SMs have been crucial in the development of antibiotics and can also act as useful compounds in the biotechnology industry. These natural products are encoded by an extensive range of biosynthetic gene clusters (BGCs). The developments in omics technologies and bioinformatic tools are contributing to a paradigm shift from traditional culturing and screening methods to bioinformatic tools and genomics to uncover BGCs that were previously unknown or transcriptionally silent. Natural product discovery using bioinformatics and omics workflow in the environment has demonstrated an extensive distribution of BGCs in various environments, such as soil, aquatic ecosystems and host microbiome environments. Computational tools provide a feasible and culture-independent route to find new secondary metabolites where traditional approaches cannot. This review will highlight some of the advances in the approaches, primarily bioinformatic, in identifying new BGCs, especially in environments where microorganisms are rarely cultured. This has allowed us to tap into the huge potential of microbial dark matter.

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