scholarly journals Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian

2021 ◽  
Vol 9 ◽  
Author(s):  
Nicole E. Avalon ◽  
Alison E. Murray ◽  
Hajnalka E. Daligault ◽  
Chien-Chi Lo ◽  
Karen W. Davenport ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Marine Invertebrates ◽  
Heterologous Gene Expression ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Combinatorial Biosynthesis ◽  
Biosynthetic Gene ◽  
Atpase Inhibitor ◽  
Synoicum Adareanum ◽  
Palmerolide A

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

scholarly journals Palmerolide PKS-NRPS gene clusters characterized from the microbiome of an Antarctic ascidian

2021 ◽  
Author(s):  
Nicole E. Avalon ◽  
Alison E. Murray ◽  
Hajnalka E. Daligault ◽  
Chien-Chi Lo ◽  
Armand E. K. Dichosa ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Marine Invertebrates ◽  
Heterologous Gene Expression ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Combinatorial Biosynthesis ◽  
Biosynthetic Gene ◽  
Atpase Inhibitor ◽  
Antarctic Species ◽  
Palmerolide A

Polyketides are a complex family of natural products that often serve competitive or pro-survival purposes but can also demonstrate bioactivity in human diseases as, for example cholesterol lowering agents, anti-infectives, or anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGC) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A putative palmerolide BGC was identified from a S. adareanum metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, and active site and motif identification were used to confirm the identity and propose the function of the 75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and multiple copies found within the metagenome-assembled genome (MAG) of Candidatus Synoicohabitans palmerolidicus. Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic species. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

scholarly journals Identification and distribution of gene clusters required for synthesis of sphingolipid metabolism inhibitors in diverse species of the filamentous fungus Fusarium

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Hye-Seon Kim ◽  
Jessica M. Lohmar ◽  
Mark Busman ◽  
Daren W. Brown ◽  
Todd A. Naumann ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Sphingolipid Metabolism ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Dehydrogenase Gene ◽  
Food And Feed ◽  
Feed Safety

Abstract Background Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs. Results Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM. Conclusion Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.

scholarly journals Characterization of the Nodularin Synthetase Gene Cluster and Proposed Theory of the Evolution of Cyanobacterial Hepatotoxins

2004 ◽  
Vol 70 (11) ◽  
pp. 6353-6362 ◽  
Author(s):  
Michelle C. Moffitt ◽  
Brett A. Neilan
Keyword(s):  
Gene Cluster ◽  
Rational Design ◽  
Polyketide Synthase ◽  
Alternative Hypothesis ◽  
Phylogenetic Analyses ◽  
Cyanobacterial Bloom ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Microcystin Synthetase

ABSTRACT Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.

Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1633-1645 ◽  
Author(s):  
Koji Ichinose ◽  
Makoto Ozawa ◽  
Keiko Itou ◽  
Kanako Kunieda ◽  
Yutaka Ebizuka
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Carrier Protein ◽  
Biosynthetic Gene ◽  
Six Genes

Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters – med and those for actinorhodin (act) and granaticin (gra) – revealed some common genes whose deduced functions are unavailable from database searches (the ‘unknowns’). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).

scholarly journals Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Lindsay K. Caesar ◽  
Matthew T. Robey ◽  
Michael Swyers ◽  
Md N. Islam ◽  
Rosa Ye ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Secondary Metabolite ◽  
Heterologous Gene Expression ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Metabolic Enzyme ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Artificial Chromosomes ◽  
Indoleamine 2,3 Dioxygenase

ABSTRACT Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster “repurposing” a primary metabolic enzyme to diversify its secondary metabolite arsenal. IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

scholarly journals Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery

2020 ◽  
Vol 48 (8) ◽  
pp. e48-e48 ◽  
Author(s):  
Peng Xu ◽  
Cyrus Modavi ◽  
Benjamin Demaree ◽  
Frederick Twigg ◽  
Benjamin Liang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Bioactive Molecules ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Plasmid Library ◽  
Polyketide Synthase Gene

Abstract Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.

scholarly journals Targeted Genome Mining—From Compound Discovery to Biosynthetic Pathway Elucidation

2020 ◽  
Vol 8 (12) ◽  
pp. 2034
Author(s):  
Nils Gummerlich ◽  
Yuriy Rebets ◽  
Constanze Paulus ◽  
Josef Zapp ◽  
Andriy Luzhetskyy
Keyword(s):  
Natural Products ◽  
Polyketide Synthase ◽  
Genomic Library ◽  
Mutational Analysis ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Feeding Experiments

Natural products are an important source of novel investigational compounds in drug discovery. Especially in the field of antibiotics, Actinobacteria have been proven to be a reliable source for lead structures. The discovery of these natural products with activity- and structure-guided screenings has been impeded by the constant rediscovery of previously identified compounds. Additionally, a large discrepancy between produced natural products and biosynthetic potential in Actinobacteria, including representatives of the order Pseudonocardiales, has been revealed using genome sequencing. To turn this genomic potential into novel natural products, we used an approach including the in-silico pre-selection of unique biosynthetic gene clusters followed by their systematic heterologous expression. As a proof of concept, fifteen Saccharothrixespanaensis genomic library clones covering predicted biosynthetic gene clusters were chosen for expression in two heterologous hosts, Streptomyceslividans and Streptomycesalbus. As a result, two novel natural products, an unusual angucyclinone pentangumycin and a new type II polyketide synthase shunt product SEK90, were identified. After purification and structure elucidation, the biosynthetic pathways leading to the formation of pentangumycin and SEK90 were deduced using mutational analysis of the biosynthetic gene cluster and feeding experiments with 13C-labelled precursors.

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

2019 ◽  
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.

Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

2021 ◽  
Vol 85 (3) ◽  
pp. 714-721
Author(s):  
Risa Takao ◽  
Katsuyuki Sakai ◽  
Hiroyuki Koshino ◽  
Hiroyuki Osada ◽  
Shunji Takahashi
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Secondary Metabolite ◽  
Response Regulator ◽  
Bac Library ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Gene Organization ◽  
Biosynthetic Gene ◽  
Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

scholarly journals Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marianna Iorio ◽  
Sahar Davatgarbenam ◽  
Stefania Serina ◽  
Paolo Criscenzo ◽  
Mitja M. Zdouc ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Systematic Investigation ◽  
Biosynthetic Gene ◽  
Wild Type ◽  
Bacterial Rna ◽  
Soil Isolate ◽  
Mutant Strains ◽  
In The Wild ◽  
Polymerase Inhibitor

AbstractWe report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

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