Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

ABSTRACTThe rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

Download Full-text

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.

Download Full-text

RRE-Finder: a Genome-Mining Tool for Class-Independent RiPP Discovery

mSystems ◽

10.1128/msystems.00267-20 ◽

2020 ◽

Vol 5 (5) ◽

Cited By ~ 1

Author(s):

Alexander M. Kloosterman ◽

Kyle E. Shelton ◽

Gilles P. van Wezel ◽

Marnix H. Medema ◽

Douglas A. Mitchell

Keyword(s):

Structure Prediction ◽

Secondary Structure Prediction ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Clusters ◽

High Confidence ◽

Genetic Feature ◽

Uniprotkb Database ◽

A Genome ◽

Mining Tool

Bioinformatics-powered discovery of novel ribosomal natural products (RiPPs) has historically been hindered by the lack of a common genetic feature across RiPP classes. Herein, we introduce RRE-Finder, a method for identifying RRE domains, which are present in a majority of prokaryotic RiPP biosynthetic gene clusters (BGCs). RRE-Finder identifies RRE domains 3,000 times faster than current methods, which rely on time-consuming secondary structure prediction. Depending on user goals, RRE-Finder can operate in precision mode to accurately identify RREs present in known RiPP classes or in exploratory mode to assist with novel RiPP discovery. Employing RRE-Finder on the UniProtKB database revealed several high-confidence RREs in novel RiPP-like clusters, suggesting that many new RiPP classes remain to be discovered.

Download Full-text

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...

Download Full-text

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.

Download Full-text

Bioinformatic and Reactivity-Based Discovery of Linaridins

10.1101/2020.07.09.196543 ◽

2020 ◽

Author(s):

Matthew A. Georgiou ◽

Shravan R. Dommaraju ◽

Xiaorui Guo ◽

Douglas A. Mitchell

Keyword(s):

Natural Product ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Clusters ◽

Road Map ◽

Streptomyces Noursei ◽

A Genome ◽

Powerful Approach ◽

Mining Tool ◽

Future Work

AbstractLinaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated threonine residue. This work describes the development of a linaridin specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored towards RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 non-redundant) linaridin biosynthetic gene clusters (BGCs). Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in dataset validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening (RBS) and isolated from Streptomyces noursei and Streptomyces auratus, respectively. RBS involves the use of a reactive chemical probe that chemoselectively modifies a functional group present in the natural product. The dehydrated amino acids present in linaridins as α/β-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4 addition using a thiol-functionalized probe. The data presented within significantly expands the number of predicted linaridin BGCs and serves as a road map for future work in the area. The combination of bioinformatics and RBS is a powerful approach to accelerate natural product discovery.

Download Full-text

Recapitulation of the evolution of biosynthetic gene clusters reveals hidden chemical diversity on bacterial genomes

10.1101/020503 ◽

2015 ◽

Cited By ~ 6

Author(s):

Pablo Cruz-Morales ◽

Christian E. Martínez-Guerrero ◽

Marco A. Morales-Escalante ◽

Luis Yáñez-Guerra ◽

Johannes Florian Kopp ◽

...

Keyword(s):

Natural Products ◽

Chemical Space ◽

Streptomyces Coelicolor ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Chemical Diversity ◽

Biosynthetic Gene ◽

Bacterial Genomes ◽

Biosynthetic Gene Clusters

AbstractNatural products have provided humans with antibiotics for millennia. However, a decline in the pace of chemical discovery exerts pressure on human health as antibiotic resistance spreads. The empirical nature of current genome mining approaches used for natural products research limits the chemical space that is explored. By integration of evolutionary concepts related to emergence of metabolism, we have gained fundamental insights that are translated into an alternative genome mining approach, termed EvoMining. As the founding assumption of EvoMining is the evolution of enzymes, we solved two milestone problems revealing unprecedented conversions. First, we report the biosynthetic gene cluster of the ‘orphan’ metabolite leupeptin in Streptomyces roseus. Second, we discover an enzyme involved in formation of an arsenic-carbon bond in Streptomyces coelicolor and Streptomyces lividans. This work provides evidence that bacterial chemical repertoire is underexploited, as well as an approach to accelerate the discovery of novel antibiotics from bacterial genomes.

Download Full-text

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

10.1101/2021.05.19.444711 ◽

2021 ◽

Author(s):

Li Cao ◽

Moshe Beiser ◽

Joseph D Koos ◽

Margarita Orlova ◽

Hader E Elashal ◽

...

Keyword(s):

Heterologous Expression ◽

Genome Mining ◽

Gene Clusters ◽

Post Translational Modification ◽

Protein Repair ◽

Lasso Peptide ◽

Lasso Peptides ◽

Modified Peptides ◽

Asparagine Deamidation

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

Download Full-text

Draft Genome Sequence and Biosynthetic Potential of the Newly Described Strain Longimicrobium terrae CB-286315T

Microbiology Resource Announcements ◽

10.1128/mra.00512-20 ◽

2020 ◽

Vol 9 (24) ◽

Author(s):

Marina Sánchez-Hidalgo ◽

Javier Pascual ◽

Ignacio González ◽

Olga Genilloud

Keyword(s):

Genome Sequence ◽

Draft Genome ◽

Gene Clusters ◽

Draft Genome Sequence ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Natural Park ◽

The Family ◽

Modified Peptides ◽

Sandwich System

ABSTRACT Longimicrobium terrae CB-286315T, the first member of the family Longimicrobiaceae, was isolated at the Sierra de Tejeda, Almijara and Alhama Natural Park, Spain, using a diffusion sandwich system (DSS). We present the draft genome sequence of this strain, which comprised 6,886,230 bp. A total of 12 putative biosynthetic gene clusters were predicted, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), polyketides, and nonribosomal peptides.

Download Full-text

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.

Download Full-text

The Draft Genome Sequence of The Endophytic Streptomyces Strain VITGV156

10.21203/rs.3.rs-791591/v1 ◽

2021 ◽

Author(s):

Veilumuthu P ◽

Nagarajan T ◽

Sasikumar S ◽

Siva R ◽

J Godwin Christopher

Keyword(s):

Secondary Metabolites ◽

Draft Genome ◽

Gc Content ◽

Gene Clusters ◽

Dominant Group ◽

Protein Coding ◽

Peptide Synthetase ◽

The Family ◽

Modified Peptides ◽

Endophytic Streptomyces

Abstract Streptomyces species is one among the dominant group of bacteria in the family Actinobacteria with a rich repertoire of secondary metabolites. Secondary metabolites with antimicrobial activity and plant growth promotor have been isolated from various Streptomyces sp. Here in this investigation, we present the draft genome of a new species, Streptomyces sp. VITGV156 isolated from healthy tomato plant (Lycopersicon esculentum) which has some rare antimicrobial secondary metabolites, like coelichelin, fluostatins, vicenistatin, nystatin, sipanmycin, and informatipeptin. The genome is 8.18 Mb in size with 6,259 protein coding genes. The average GC content of the genome is 72.61 %. Preliminary analysis with antiSMASH 6.0 revealed the presence of 29 biosynthetic gene clusters for the synthesis of potential secondary metabolites. These includes 4 NRPS (non – ribosomal peptide synthetase), 7 PKS (Polyketide Synthases), 2 RiPP (Ribosomally synthesized and post-translationally modified peptides) clusters. When we look into genes associated with secondary metabolites, 406 genes are present which includes 184 genes for cofactor and vitamins, 72 genes for terpenoids and polyketides, 70 genes for xenobiotics and 80 genes for other metabolites are present. Comparative genome analysis of VITGV156 with its closest neighbor Streptomyces luteus strain TRM45540 revealed ANI 91.22% and dDDH value 44.00%.

Download Full-text