scholarly journals Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors

2020 ◽  
Author(s):  
Dina Kačar ◽  
Librada M Cañedo ◽  
Pilar Rodríguez ◽  
Elena Gonzalez ◽  
Beatriz Galán ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Marine Bacteria ◽  
Genome Mining ◽  
Evolutionary Relationship ◽  
Gene Clusters ◽  
Comparative Genomic ◽  
Homologous Gene ◽  
Modular Architecture ◽  
Orphan Gene ◽  
Strong Antitumor Activity

AbstractGlutaramide-containing polyketides are known as potent antitumoral and antimetastatic agents. However, the associated gene clusters have only been identified and studied in a few Streptomyces producers and sole Burkholderia gladioli symbiont. The new glutaramide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster, and the sesbanimide modular assembly is proposed. Interestingly, numerous homologous orphan gene clusters were localized in distantly related bacteria and used as comparative genomic assets for a more global characterization of sbn like-clusters. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologs in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutaramide-like and pederin-like pathways, as well as the functionality of their synthetic products.SignificanceGlutaramide-containing polyketides are still a largely understudied group of polyketides, produced mainly by the genera Streptomyces, with a great potential for antitumor drug production. Here, we describe genomes of two cultivable marine bacteria, Stappia indica PHM037 and Labrenzia aggregata PHM038, producers of the cytotoxic glutaramide-family polyketides sesbanimide A and C with chemical elucidation of newly identified analogs D, E and F. Genome mining revealed trans-AT PKS gene cluster responsible for sesbanimide biosynthesis. Although there are numerous homologous gene clusters present in remarkably different bacteria, this is the first time that the biosynthesis product has been reported. The comparative genome analysis reveals stunning, cryptic evolutionary relationship between sesbanimides, glutaramides from Streptomyces spp. and the pederin-family gene clusters.

scholarly journals Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
High Performance ◽  
Pathogenic Bacteria ◽  
Genome Mining ◽  
Gene Clusters ◽  
Von Willebrand Disease ◽  
Biosynthesis Gene Cluster ◽  
Content Type ◽  
Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

scholarly journals Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

2020 ◽  
Vol 11 ◽  
Author(s):  
Jin Lü ◽  
Qingshan Long ◽  
Zhilong Zhao ◽  
Lu Chen ◽  
Weijun He ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Bac Library ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Saccharopolyspora Erythraea ◽  
Heterologous Production ◽  
Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

scholarly journals Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

2009 ◽  
Vol 76 (1) ◽  
pp. 283-293 ◽  
Author(s):  
Hanne Jørgensen ◽  
Kristin F. Degnes ◽  
Alexander Dikiy ◽  
Espen Fjærvik ◽  
Geir Klinkenberg ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Evolutionary Relationship ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Side Chain ◽  
Small Ring ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
The Difference ◽  
High Level

ABSTRACT A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the β-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis.

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 Odorant-binding proteins in canine anal sac glands indicate an evolutionarily conserved role in mammalian chemical communication

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sunita Janssenswillen ◽  
Kim Roelants ◽  
Sebastien Carpentier ◽  
Hilde de Rooster ◽  
Mieke Metzemaekers ◽  
...  
Keyword(s):  
Chemical Communication ◽  
Binding Proteins ◽  
Evolutionary Relationship ◽  
Gene Clusters ◽  
Pseudoautosomal Region ◽  
Comparative Genomic ◽  
Odorant Binding Proteins ◽  
Wide Range ◽  
Anal Sac ◽  
Odorant Binding

Abstract Background Chemical communication is an important aspect of the behavioural ecology of a wide range of mammals. In dogs and other carnivores, anal sac glands are thought to convey information to conspecifics by secreting a pallet of small volatile molecules produced by symbiotic bacteria. Because these glands are unique to carnivores, it is unclear how their secretions relate to those of other placental mammals that make use of different tissues and secretions for chemical communication. Here we analyse the anal sac glands of domestic dogs to verify the secretion of proteins and infer their evolutionary relationship to those involved in the chemical communication of non-carnivoran mammals. Results Proteomic analysis of anal sac gland secretions of 17 dogs revealed the consistently abundant presence of three related proteins. Homology searches against online databases indicate that these proteins are evolutionary related to ‘odorant binding proteins’ (OBPs) found in a wide range of mammalian secretions and known to contribute to chemical communication. Screening of the dog’s genome sequence show that the newly discovered OBPs are encoded by a single cluster of three genes in the pseudoautosomal region of the X-chromosome. Comparative genomic screening indicates that the same locus is shared by a wide range of placental mammals and that it originated at least before the radiation of extant placental orders. Phylogenetic analyses suggest a dynamic evolution of gene duplication and loss, resulting in large gene clusters in some placental taxa and recurrent loss of this locus in others. The homology of OBPs in canid anal sac glands and those found in other mammalian secretions implies that these proteins maintained a function in chemical communication throughout mammalian evolutionary history by multiple shifts in expression between secretory tissues involved in signal release and nasal mucosa involved in signal reception. Conclusions Our study elucidates a poorly understood part of the biology of a species that lives in close association with humans. In addition, it shows that the protein repertoire underlying chemical communication in mammals is more evolutionarily stable than the variation of involved glands and tissues would suggest.

scholarly journals FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution

2021 ◽  
Vol 17 (9) ◽  
pp. e1009372
Author(s):  
Gabriel A. Vignolle ◽  
Denise Schaffer ◽  
Leopold Zehetner ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner ◽  
...  
Keyword(s):  
Food Additives ◽  
Protein A ◽  
Genome Mining ◽  
Gene Clusters ◽  
Essential Genes ◽  
Comparative Genomic ◽  
Automated Identification ◽  
Gap Genes ◽  
Automated Method ◽  
Proteome Database

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

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

2017 ◽  
Vol 114 (52) ◽  
pp. E11121-E11130 ◽  
Author(s):  
Gregory C. A. Amos ◽  
Takayoshi Awakawa ◽  
Robert N. Tuttle ◽  
Anne-Catrin Letzel ◽  
Min Cheol Kim ◽  
...  
Keyword(s):  
Natural Product ◽  
Gene Cluster ◽  
Genetic Manipulation ◽  
Expression Profiles ◽  
Genome Mining ◽  
Gene Clusters ◽  
Comparative Transcriptomics ◽  
Regulatory Control ◽  
Biosynthetic Gene ◽  
Metabolomics Data

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.

scholarly journals Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria

2010 ◽  
Vol 77 (4) ◽  
pp. 1214-1220 ◽  
Author(s):  
Toshiki Furuya ◽  
Satomi Hirose ◽  
Hisashi Osanai ◽  
Hisashi Semba ◽  
Kuniki Kino
Keyword(s):  
Gene Cluster ◽  
Large Subunit ◽  
Gene Clusters ◽  
Small Subunit ◽  
Homologous Gene ◽  
Oxidation Activity ◽  
Monooxygenase Gene ◽  
Regioselective Oxidation ◽  
Binuclear Iron ◽  
Induced Proteins

ABSTRACTMycobacterium goodiistrain 12523 is an actinomycete that is able to oxidize phenol regioselectively at theparaposition to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity ofM. goodiistrain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 inMycobacterium smegmatisstrain mc2155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster ofM. smegmatisstrain mc2155 and its homologous gene cluster found inM. goodiistrain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designatedmimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When themimAgene (Msmeg_1971) ofM. smegmatisstrain mc2155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of themimAgene ofM. smegmatisstrain mc2155 or ofM. goodiistrain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

scholarly journals Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper

2021 ◽  
Author(s):  
Alexander G McFarland ◽  
Nolan W Kennedy ◽  
Carolyn E Mills ◽  
Danielle Tullman-Ercek ◽  
Curtis Huttenhower ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Population Level ◽  
Gene Clusters ◽  
Homologous Gene ◽  
Integrative And Conjugative Elements ◽  
Density Based Clustering ◽  
Command Line Tool ◽  
Conserved Gene ◽  
Similar Gene

Motivation: Identifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. Results: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster in 435 genomes containing mixed taxa. In a subsequent application investigating the diversity and impact of gene complete and incomplete LT2 Pdu gene clusters in 1130 S. enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements.

scholarly journals A Pseudoalteromonas clade with remarkable biosynthetic potential

2021 ◽  
Author(s):  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Jesse Cain ◽  
John A. Kalaitzis ◽  
Brett A. Neilan
Keyword(s):  
Natural Products ◽  
Gene Cluster ◽  
Genome Mining ◽  
Gene Clusters ◽  
Average Density ◽  
Biologically Active ◽  
Biosynthesis Gene Cluster ◽  
Diverse Range ◽  
Siderophore Biosynthesis ◽  
Biosynthesis Gene

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here we report the biochemical and genomic analysis of Pseudoalteromonas sp. HM-SA03, isolated from the blue-ringed octopus, Hapalochalaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides and four hitherto novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of ten biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the under-explored potential of Pseudoalteromonas as a source of new natural products. Importance This study demonstrates that the Pseudoalteromonas strain, HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We have identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we have demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

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