scholarly journals Transporter Gene-mediated Typing for Detection and Genome Mining of Lipopeptide-producing Pseudomonas

2021 ◽  
Author(s):  
Léa Girard ◽  
Niels Geudens ◽  
Brent Pauwels ◽  
Monica Höfte ◽  
José C. Martins ◽  
...  
Keyword(s):  
Genome Mining ◽  
Chemical Characterization ◽  
Gene Clusters ◽  
Amplicon Sequencing ◽  
Chemical Diversity ◽  
Ecological Niches ◽  
Transporter Gene ◽  
A Genome ◽  
Wide Range ◽  
Suitable Target

Pseudomonas lipopeptides (LPs) are involved in diverse ecological functions and have biotechnological potential associated with their antimicrobial and/or anti-proliferative activities. They are synthesized by multi-modular non-ribosomal peptide synthetases which, together with transport and regulatory proteins, are encoded by large biosynthetic gene clusters (BGCs). These secondary metabolites are classified in distinct families based on sequence and length of the oligopeptide, and size of the macrocycle, if present. Phylogeny of PleB, the MacB-like transporter that is part of a dedicated ATP-dependent tripartite efflux system driving export of Pseudomonas LPs, revealed a strong correlation with LP chemical diversity. As each LP BGC carries its cognate pleB , PleB is suitable as a diagnostic sequence for genome mining, allowing assignment of the putative metabolite to a particular LP family. In addition, pleB proved a suitable target gene for an alternative PCR method to detect LP-producing Pseudomonas , not relying on amplification of catalytic domains of the biosynthetic enzymes. Combined with amplicon sequencing, this approach enabled typing of Pseudomonas strains as potential producers of a LP belonging to one of ten different families, underscoring its value for strain prioritization. This was validated by chemical characterization of known LPs from three different families secreted by novel producers isolated from the rice or maize rhizosphere, namely the type strains of Pseudomonas fulva (putisolvin), Pseudomonas zeae (tensin) and Pseudomonas xantholysinigenes (xantholysin). In addition, a new member of the Bananamide family, prosekin, was discovered in the type strain of Pseudomonas prosekii , an Antarctic isolate. Importance Pseudomonas are ubiquitous bacteria able to thrive in a wide range of ecological niches and lipopeptides often support their lifestyle but also their interaction with other micro- and macro-organisms. Therefore, the production of lipopeptides is widespread among Pseudomonas strains. Consequently, Pseudomonas lipopeptide research affects not only chemists and microbiologists but touches a much broader audience, including biochemists, ecologists and plant biologists. In this study we present a reliable transporter gene-guided approach for the detection and/or typing of Pseudomonas lipopeptide producers. Indeed, it allows to readily assess the lipopeptide diversity among sets of Pseudomonas isolates and differentiate strains likely to produce known lipopeptides from producers of potentially novel lipopeptides. This work provides a valuable tool that can also be integrated in a genome mining strategy and adapted for the typing of other specialized metabolites.

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

2018 ◽  
Author(s):  
Javier Santos-Aberturas ◽  
Govind Chandra ◽  
Luca Frattaruolo ◽  
Rodney Lacret ◽  
Thu H. Pham ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Bioactive Molecules ◽  
Post Translational Modification ◽  
Biosynthetic Gene Clusters ◽  
New Family ◽  
The Family ◽  
Modified Peptides ◽  
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.

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

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
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.

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

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.

scholarly journals Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jingyan Zhang ◽  
Ying Sun ◽  
Yeji Wang ◽  
Xin Chen ◽  
Lu Xue ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Scale Up ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Wild Type ◽  
Aromatic Polyketides ◽  
A Genome

Abstract Background Rubiginones belong to the angucycline family of aromatic polyketides, and they have been shown to potentiate the vincristine (VCR)-induced cytotoxicity against VCR-resistant cancer cell lines. However, the biosynthetic gene clusters (BGCs) and biosynthetic pathways for rubiginones have not been reported yet. Results In this study, based on bioinformatics analysis of the genome of Streptomyces sp. CB02414, we predicted the functions of the two type II polyketide synthases (PKSs) BGCs. The rub gene cluster was predicted to encode metabolites of the angucycline family. Scale-up fermentation of the CB02414 wild-type strain led to the discovery of eight rubiginones, including five new ones (rubiginones J, K, L, M, and N). Rubiginone J was proposed to be the final product of the rub gene cluster, which features extensive oxidation on the A-ring of the angucycline skeleton. Based on the production profiles of the CB02414 wild-type and the mutant strains, we proposed a biosynthetic pathway for the rubiginones in CB02414. Conclusions A genome mining strategy enabled the efficient discovery of new rubiginones from Streptomyces sp. CB02414. Based on the isolated biosynthetic intermediates, a plausible biosynthetic pathway for the rubiginones was proposed. Our research lays the foundation for further studies on the mechanism of the cytochrome P450-catalyzed oxidation of angucyclines and for the generation of novel angucyclines using combinatorial biosynthesis strategies.

scholarly journals Activation and enhancement of Caerulomycin A biosynthesis in marine-derived Actinoalloteichus sp. AHMU CJ021 by combinatorial genome mining strategies

2020 ◽  
Author(s):  
Yunchang Xie ◽  
Jiawen Chen ◽  
Bo Wang ◽  
Tai Chen ◽  
Junyu Chen ◽  
...  
Keyword(s):  
Natural Products ◽  
Mutant Strain ◽  
Genome Mining ◽  
Gene Clusters ◽  
Immunosuppressive Drug ◽  
Biosynthetic Gene ◽  
Bioactive Natural Products ◽  
Drug Lead ◽  
A Genome ◽  
Mining Work

Abstract Backgrounds: Activation of silent biosynthetic gene clusters (BGCs) in marine-derived actinomycete strains is a feasible strategy to discover bioactive natural products. Actinoalloteichus sp. AHMU CJ021, isolated from the seashore, was shown to contain an intact but silent caerulomycin A (CRM A) BGC-cam in its genome. Thus, a genome mining work was preformed to activate the strain’s bioproduction of CRM A, an immunosuppressive drug lead with diverse bioactivities.Results: To well activate the expression of cam, ribosomal engineering was adopted to treat the wild type Actinoalloteichus sp. AHMU CJ021. The initial mutant strain XC-11G with gentamycin resistance and CRM A bioproduction titer of 42.51 ± 4.22 mg/L was selected from all generated mutant strains by gene expression comparison of the essential biosynthetic gene-camE. The titer of CRM A bioproduction was then improved by two strain breeding methods via UV mutagenesis and cofactor engineering-directed increasing of intracellular riboflavin, which finally generated the optimal mutant strain XC-11GUR with a CRM A bioproduction titer of 113.91 ± 7.58 mg/L. Subsequently, this titer of strain XC-11GUR was improved to 618.61 ± 16.29 mg/L through medium optimization together with further adjustment derived from response surface methodology. In terms of this 14.7 folds increase in the titer of CRM A compared to the initial value, strain XC-GUR could be a well alternative strain for CRM A development.Conclusions: Our results have constructed an ideal CRM A producer. More importantly, our efforts also have demonstrated the effectiveness of abovementioned combinatorial strategies, which is applicable to the genome mining of bioactive natural products from abundant actinomycetes strains.

scholarly journals A highly contiguous genome assembly of the bat hawkmoth Hyles vespertilio (Lepidoptera: Sphingidae)

GigaScience ◽  
2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Martin Pippel ◽  
David Jebb ◽  
Franziska Patzold ◽  
Sylke Winkler ◽  
Heiko Vogel ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Gene Annotation ◽  
Incomplete Lineage Sorting ◽  
Read Length ◽  
Ecological Niches ◽  
Average Read Length ◽  
General Applicability ◽  
Pacific Biosciences ◽  
A Genome ◽  
Wide Range

Abstract Background Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation. Results Here we present a high-quality genome assembly of the bat hawkmoth Hyles vespertilio, the first reference genome of a member of the Hyles genus. We generated 51× Pacific Biosciences long reads with an average read length of 8.9 kb. Pacific Biosciences reads longer than 4 kb were assembled into contigs, resulting in a 651.4-Mb assembly consisting of 530 contigs with an N50 value of 7.5 Mb. The circular mitochondrial contig has a length of 15,303 bp. The H. vespertilio genome is very repeat-rich and exhibits a higher repeat content (50.3%) than other Bombycoidea species such as Bombyx mori (45.7%) and Manduca sexta (27.5%). We developed a comprehensive gene annotation workflow to obtain consensus gene models from different evidence including gene projections, protein homology, transcriptome data, and ab initio predictions. The resulting gene annotation is highly complete with 94.5% of BUSCO genes being completely present, which is higher than the BUSCO completeness of the B. mori (92.2%) and M. sexta (90%) annotations. Conclusions Our gene annotation strategy has general applicability to other genomes, and the H. vespertilio genome provides a valuable molecular resource to study a range of questions in this genus, including phylogeny, incomplete lineage sorting, speciation, and hybridization. A genome browser displaying the genome, alignments, and annotations is available at https://genome-public.pks.mpg.de/cgi-bin/hgTracks?db=HLhylVes1.

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

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...

scholarly journals Discovery of an Antibiotic-Related Small Protein of Biocontrol Strain Pseudomonas sp. Os17 by a Genome-Mining Strategy

2020 ◽  
Vol 11 ◽  
Author(s):  
Kasumi Takeuchi ◽  
Wataru Tsuchiya ◽  
Zui Fujimoto ◽  
Kosumi Yamada ◽  
Nobutaka Someya ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genome Mining ◽  
Signal Transduction Pathway ◽  
Antibiotic Activity ◽  
The Novel ◽  
Post Translational Modifications ◽  
A Genome ◽  
Genomic Study ◽  
Wide Range ◽  
Biocontrol Strain

Many root-colonizing Pseudomonas spp. exhibiting biocontrol activities produce a wide range of secondary metabolites that exert antibiotic effects against other microbes, nematodes, and insects in the rhizosphere. The expression of these secondary metabolites depends on the Gac/Rsm signal transduction pathway. Based on the findings of a previous genomic study on newly isolated biocontrol pseudomonad strains, we herein investigated the novel gene cluster OS3, which consists of four genes (Os1348–Os1351) that are located upstream of putative efflux transporter genes (Os1352–Os1355). Os1348 was predicted to encode an 85-aa small precursor protein, the expression of which was under the control of GacA, and an X-ray structural analysis suggested that the Os1348 protein formed a dimer. The mutational loss of the Os1348 gene decreased the antibiotic activity of Pseudomonas sp. Os17 without changing its growth rate. The Os1349–1351 genes were predicted to be involved in post-translational modifications. Intracellular levels of the Os1348 protein in the deficient mutant of each gene differed from that in wild-type cells. These results suggest that Os1348 is involved in antibiotic activity and that the structure or expression of this protein is under the control of downstream gene products.

scholarly journals Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A

2022 ◽  
Vol 12 ◽  
Author(s):  
Joseph Wambui ◽  
Marc J. A. Stevens ◽  
Simon Sieber ◽  
Nicole Cernela ◽  
Vincent Perreten ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Proteolytic Enzymes ◽  
Genome Mining ◽  
Gene Clusters ◽  
Public Health Issue ◽  
Ecological Niches ◽  
Primary Screening ◽  
Secondary Screening ◽  
Natural Variant ◽  
Screening Assays

Antimicrobial resistance in pathogenic bacteria is considered a major public health issue necessitating the discovery of alternative antimicrobial compounds. In this regard, targeted genome mining in bacteria occupying under-explored ecological niches has the potential to reveal such compounds, including bacteriocins. In this study, we determined the bacteriocin biosynthetic potential of the psychrophilic Clostridium estertheticum complex (CEC) through a combination of genome mining and phenotypic screening assays. The genome mining was performed in 40 CEC genomes using antiSMASH. The production of bacteriocin-like compounds was phenotypically validated through agar well (primary screening) and disk diffusion (secondary screening) assays using cell free supernatants (CFS) and partially purified extracts, respectively. Stability of four selected CFS against proteolytic enzymes, temperature and pH was determined while one CFS was analyzed by HRMS and MS/MS to identify potential bacteriocins. Twenty novel bacteriocin biosynthetic gene clusters (BBGC), which were classified into eight (six lantibiotics and two sactipeptides) distinct groups, were discovered in 18 genomes belonging to C. estertheticum (n = 12), C. tagluense (n = 3) and genomospecies2 (n = 3). Primary screening linked six BBGC with narrow antimicrobial activity against closely related clostridia species. All four preselected CFS retained activity after exposure to different proteolytic, temperature and pH conditions. Secondary screening linked BBGC1 and BBGC7 encoding a lantibiotic and sactipeptide, respectively, with activity against Bacillus cereus while lantibiotic-encoding BBGC2 and BBGC3 were linked with activity against B. cereus, Staphylococcus aureus (methicillin-resistant), Escherichia coli and Pseudomonas aeruginosa. MS/MS analysis revealed that C. estertheticum CF004 produces cesin A, a short natural variant of nisin, and HRMS indicated the production of a novel sactipeptide named estercticin A. Therefore, we have shown the CEC, in particular C. estertheticum, is a source of novel and stable bacteriocins that have activities against clinically relevant pathogens.

scholarly journals Global Genome Mining Reveals the Distribution of Diverse Thioamidated RiPP Biosynthesis Gene Clusters

2021 ◽  
Vol 12 ◽  
Author(s):  
Jessie James Limlingan Malit ◽  
Chuanhai Wu ◽  
Ling-Li Liu ◽  
Pei-Yuan Qian
Keyword(s):  
Protein Pair ◽  
Genome Mining ◽  
Gene Clusters ◽  
Cytotoxic Activities ◽  
Related Gene ◽  
Peptide Backbone ◽  
Precursor Peptide ◽  
Wide Range ◽  
Biosynthesis Gene ◽  
Modified Peptides

Thioamidated ribosomally synthesized and post-translationally modified peptides (RiPPs) are recently characterized natural products with wide range of potent bioactivities, such as antibiotic, antiproliferative, and cytotoxic activities. These peptides are distinguished by the presence of thioamide bonds in the peptide backbone catalyzed by the YcaO-TfuA protein pair with its genes adjacent to each other. Genome mining has facilitated an in silico approach to identify biosynthesis gene clusters (BGCs) responsible for thioamidated RiPP production. In this work, publicly available genomic data was used to detect and illustrate the diversity of putative BGCs encoding for thioamidated RiPPs. AntiSMASH and RiPPER analysis identified 613 unique TfuA-related gene cluster families (GCFs) and 797 precursor peptide families, even on phyla where the presence of these clusters have not been previously described. Several additional biosynthesis genes are colocalized with the detected BGCs, suggesting an array of possible chemical modifications. This study shows that thioamidated RiPPs occupy a widely unexplored chemical landscape.

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