Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality

Bacterial natural products remain an important source of new medicines. DNA sequencing has revealed that a majority of natural product biosynthetic gene clusters (BGCs) maintained in bacterial genomes have yet to be linked to the small molecules whose biosynthesis they encode. Efforts to discover the products of these orphan BGCs are driving the development of genome mining techniques based on the premise that many are transcriptionally silent during normal laboratory cultivation. Here, we employ comparative transcriptomics to assess BGC expression among four closely related strains of marine bacteria belonging to the genusSalinispora. The results reveal that slightly more than half of the BGCs are expressed at levels that should facilitate product detection. By comparing the expression profiles of similar gene clusters in different strains, we identified regulatory genes whose inactivation appears linked to cluster silencing. The significance of these subtle differences between expressed and silent BGCs could not have been predicted a priori and was only revealed by comparative transcriptomics. Evidence for the conservation of silent clusters among a larger number of strains for which genome sequences are available suggests they may be under different regulatory control from the expressed forms or that silencing may represent an underappreciated mechanism of gene cluster evolution. Coupling gene expression and metabolomics data established a bioinformatic link between the salinipostins and their associated BGC, while genetic manipulation established the genetic basis for this series of compounds, which were previously unknown fromSalinispora pacifica.

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A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators

mBio ◽

10.1128/mbio.01230-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 6

Author(s):

Loïc Martinet ◽

Aymeric Naômé ◽

Benoit Deflandre ◽

Marta Maciejewska ◽

Déborah Tellatin ◽

...

Keyword(s):

Natural Product ◽

Gene Cluster ◽

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Bioactive Molecules ◽

Bioactive Metabolites ◽

Biosynthetic Gene ◽

Single Family ◽

Biosynthetic Gene Clusters

ABSTRACT Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions. IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

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Comparative Genomics and Metabolomics in the Genus Nocardia

mSystems ◽

10.1128/msystems.00125-20 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 2

Author(s):

Daniel Männle ◽

Shaun M. K. McKinnie ◽

Shrikant S. Mantri ◽

Katharina Steinke ◽

Zeyin Lu ◽

...

Keyword(s):

Natural Product ◽

Gene Cluster ◽

Gene Clusters ◽

Chemical Diversity ◽

Biosynthetic Gene ◽

Compound Identification ◽

Biosynthetic Gene Clusters ◽

Metabolomics Data ◽

Similarity Networks ◽

Gene Similarity

ABSTRACT Using automated genome analysis tools, it is often unclear to what degree genetic variability in homologous biosynthetic pathways relates to structural variation. This hampers strain prioritization and compound identification and can lead to overinterpretation of chemical diversity. Here, we assessed the metabolic potential of Nocardia, an underinvestigated actinobacterial genus that is known to comprise opportunistic human pathogens. Our analysis revealed a plethora of putative biosynthetic gene clusters of various classes, including polyketide, nonribosomal peptide, and terpenoid pathways. Furthermore, we used the highly conserved biosynthetic pathway for nocobactin-like siderophores to investigate how gene cluster differences correlate to structural differences in the produced compounds. Sequence similarity networks generated by BiG-SCAPE (Biosynthetic Gene Similarity Clustering and Prospecting Engine) showed the presence of several distinct gene cluster families. Metabolic profiling of selected Nocardia strains using liquid chromatography-mass spectrometry (LC-MS) metabolomics data, nuclear magnetic resonance (NMR) spectroscopy, and GNPS (Global Natural Product Social molecular networking) revealed that nocobactin-like biosynthetic gene cluster (BGC) families above a BiG-SCAPE threshold of 70% can be assigned to distinct structural types of nocobactin-like siderophores. IMPORTANCE Our work emphasizes that Nocardia represent a prolific source for natural products rivaling better-characterized genera such as Streptomyces or Amycolatopsis. Furthermore, we showed that large-scale analysis of biosynthetic gene clusters using similarity networks with high stringency allows the distinction and prediction of natural product structural variations. This will facilitate future genomics-driven drug discovery campaigns.

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Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽

10.3390/biom10050734 ◽

2020 ◽

Vol 10 (5) ◽

pp. 734 ◽

Cited By ~ 2

Author(s):

Yawei Zhao ◽

Guoquan Li ◽

Yunliang Chen ◽

Yinhua Lu

Keyword(s):

Natural Product ◽

Genome Editing ◽

Genetic Manipulation ◽

Chemical Compounds ◽

Gene Clusters ◽

Future Research ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Research Focus ◽

Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

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Genomic Assemblies of Members of Burkholderia and Related Genera as a Resource for Natural Product Discovery

Microbiology Resource Announcements ◽

10.1128/mra.00485-20 ◽

2020 ◽

Vol 9 (42) ◽

Author(s):

Alex J. Mullins ◽

Cerith Jones ◽

Matthew J. Bull ◽

Gordon Webster ◽

Julian Parkhill ◽

...

Keyword(s):

Natural Product ◽

Genome Mining ◽

Genomic Analysis ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Natural Product Discovery

ABSTRACT The genomes of 450 members of Burkholderiaceae, isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

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Whole-genome sequence of bioactive streptomycete derived from mangrove forest in Malaysia, Streptomyces sp. MUSC 14

Progress In Microbes & Molecular Biology ◽

10.36877/pmmb.a0000195 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Hooi-Leng Ser ◽

Loh Teng-Hern Tan ◽

Wen-Si Tan ◽

Wai-Fong Yin ◽

Kok-Gan Chan

Keyword(s):

East Coast ◽

Genetic Manipulation ◽

Genome Mining ◽

Gene Clusters ◽

Peninsular Malaysia ◽

Whole Genome Sequence ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Streptomyces Sp ◽

A Genome

The contribution of streptomycetes to human health is undeniably important and significant, given that these filamentous microbes can produce interesting compounds that can be used to cure deadly infections and even cancer. Isolated from the east coast of Peninsular Malaysia, Streptomyces sp. MUSC 14 has shown significant antioxidant capacity. The current study explores the genomic potential of MUSC 14 via a genome mining approach. The genome size of MUSC 14 is 10,274,825 bp with G + C content of 71.3 %. AntiSMASH analysis revealed a total of nine biosynthetic gene clusters (with more than 80 % similarities to known gene clusters). This information serves as an important foundation for subsequent studies, particularly the purification and isolation of bioactive compounds by genetic manipulation techniques.

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Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Chemical Science ◽

10.1039/d1sc01456k ◽

2021 ◽

Author(s):

Alicia H Russell ◽

Natalia Miguel Vior ◽

Edward Steven Hems ◽

Rodney Lacret ◽

Andrew William Truman

Keyword(s):

Natural Product ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Wide Range ◽

Modified Peptides

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is...

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Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

10.1101/2020.05.04.076059 ◽

2020 ◽

Author(s):

Alicia H. Russell ◽

Natalia M. Vior ◽

Edward S. Hems ◽

Rodney Lacret ◽

Andrew W. Truman

Keyword(s):

Natural Product ◽

Genome Mining ◽

Gene Clusters ◽

Model Organisms ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Post Translational Modifications ◽

Streptomyces Albus ◽

Modified Peptides ◽

Mining Tool

ABSTRACTRibosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor detection of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel, diverse and highly conserved family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albus J1074 was characterised, leading to the discovery of streptamidine, a novel-amidine containing RiPP. This highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

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BiG-SLiCE: A Highly Scalable Tool Maps the Diversity of 1.2 Million Biosynthetic Gene Clusters

10.1101/2020.08.17.240838 ◽

2020 ◽

Cited By ~ 3

Author(s):

Satria A. Kautsar ◽

Justin J. J. van der Hooft ◽

Dick de Ridder ◽

Marnix H. Medema

Keyword(s):

Natural Product ◽

Biological Activities ◽

Genome Mining ◽

Gene Clusters ◽

Genomic Diversity ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Microbial Genomes ◽

Natural Product Discovery ◽

User Friendly

AbstractBackgroundGenome mining for Biosynthetic Gene Clusters (BGCs) has become an integral part of natural product discovery. The >200,000 microbial genomes now publicly available hold information on abundant novel chemistry. One way to navigate this vast genomic diversity is through comparative analysis of homologous BGCs, which allows identification of cross-species patterns that can be matched to the presence of metabolites or biological activities. However, current tools suffer from a bottleneck caused by the expensive network-based approach used to group these BGCs into Gene Cluster Families (GCFs).ResultsHere, we introduce BiG-SLiCE, a tool designed to cluster massive numbers of BGCs. By representing them in Euclidean space, BiG-SLiCE can group BGCs into GCFs in a non-pairwise, near-linear fashion. We used BiG-SLiCE to analyze 1,225,071 BGCs collected from 209,206 publicly available microbial genomes and metagenome-assembled genomes (MAGs) within ten days on a typical 36-cores CPU server. We demonstrate the utility of such analyses by reconstructing a global map of secondary metabolic diversity across taxonomy to identify uncharted biosynthetic potential. BiG-SLiCE also provides a "query mode" that can efficiently place newly sequenced BGCs into previously computed GCFs, plus a powerful output visualization engine that facilitates user-friendly data exploration.ConclusionsBiG-SLiCE opens up new possibilities to accelerate natural product discovery and offers a first step towards constructing a global, searchable interconnected network of BGCs. As more genomes get sequenced from understudied taxa, more information can be mined to highlight their potentially novel chemistry. BiG-SLiCE is available via https://github.com/medema-group/bigslice.

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Genome Mining and Metabolomics Uncover a Rare d-Capreomycidine Containing Natural Product and Its Biosynthetic Gene Cluster

ACS Chemical Biology ◽

10.1021/acschembio.0c00663 ◽

2020 ◽

Vol 15 (11) ◽

pp. 3013-3020

Author(s):

James H. Tryon ◽

Jennifer C. Rote ◽

Li Chen ◽

Matthew T. Robey ◽

Marvin M. Vega ◽

...

Keyword(s):

Natural Product ◽

Gene Cluster ◽

Genome Mining ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene

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Genome mining and UHPLC–QTOF–MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2

BMC Genomics ◽

10.1186/s12864-020-07160-2 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Zhenhe Su ◽

Xiuye Chen ◽

Xiaomeng Liu ◽

Qinggang Guo ◽

Shezeng Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Gene Cluster ◽

Biological Activities ◽

Genome Mining ◽

Gene Clusters ◽

Integrative Approach ◽

Antimicrobial Compounds ◽

Biosynthetic Gene ◽

Similar Function ◽

Biosynthetic Gene Clusters

Abstract Background Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities. Results Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. Conclusions Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC–QTOF–MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

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