scholarly journals Biosynthesis of alkylcitric acids in Aspergillus niger involves both co-localized and unlinked genes

2019 ◽  
Author(s):  
Sylvester Palys ◽  
Thi Thanh My Pham ◽  
Adrian Tsang
Keyword(s):  
Aspergillus Niger ◽  
Biosynthetic Pathway ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Phylogenomic Analysis ◽  
Total Production ◽  
Regulator Gene ◽  
Transcription Regulator ◽  
Overexpression Strain ◽  
Alkyl Tail

AbstractFilamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl “tail” and a citrate-derived “head”. We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from a litre of culture filtrate 8.5 grams of extract containing seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised ~ 95% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed an in-cluster cis-aconitate decarboxylase gene (cadA) in A. oryzae and A. flavus, which in A. niger is located on a different chromosome. Overexpression of the A. niger cadA and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

scholarly journals Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

2002 ◽  
Vol 68 (6) ◽  
pp. 2885-2892 ◽  
Author(s):  
Rosie E. Bradshaw ◽  
Deepak Bhatnagar ◽  
Rebecca J. Ganley ◽  
Carmel J. Gillman ◽  
Brendon J. Monahan ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Structural Similarity ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Hybridization Probe ◽  
Forest Pathogen ◽  
Major Facilitator ◽  
Dothistroma Pini

ABSTRACT Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.

scholarly journals Unexpected distribution of the 4-formylaminooxyvinylglycine (FVG) biosynthetic pathway in Pseudomonas and beyond

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0247348
Author(s):  
Edward W. Davis ◽  
Rachel A. Okrent ◽  
Viola A. Manning ◽  
Kristin M. Trippe
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Species Group ◽  
Biosynthetic Gene ◽  
Additional Gene ◽  
Genetic Potential ◽  
Lines Of Evidence

The biological herbicide and antibiotic 4-formylaminooxyvinylglycine (FVG) was originally isolated from several rhizosphere-associated strains of Pseudomonas fluorescens. Biosynthesis of FVG is dependent on the gvg biosynthetic gene cluster in P. fluorescens. In this investigation, we used comparative genomics to identify strains with the genetic potential to produce FVG due to presence of a gvg gene cluster. These strains primarily belong to two groups of Pseudomonas, P. fluorescens and P. syringae, however, a few strains with the gvg cluster were found outside of Pseudomonas. Mass spectrometry confirmed that all tested strains of the P. fluorescens species group produced FVG. However, P. syringae strains did not produce FVG under standard conditions. Several lines of evidence regarding the transmission of the gvg cluster including a robust phylogenetic analysis suggest that it was introduced multiple times through horizontal gene transfer within the Pseudomonas lineage as well as in select lineages of Thiomonas, Burkholderia and Pantoea. Together, these data broaden our understanding of the evolution and diversity of FVG biosynthesis. In the course of this investigation, additional gene clusters containing only a subset of the genes required to produce FVG were identified in a broad range of bacteria, including many non-pseudomonads.

scholarly journals Unexpected distribution of the 4-formylaminooxyvinylglycine (FVG) biosynthetic pathway in Pseudomonas and beyond

2021 ◽  
Author(s):  
Edward W. Davis ◽  
Rachel A. Okrent ◽  
Viola A. Manning ◽  
Kristin M. Trippe
Keyword(s):  
Mass Spectrometry ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Species Group ◽  
Biosynthetic Gene ◽  
Additional Gene ◽  
Genetic Potential ◽  
Lines Of Evidence

ABSTRACTThe biological herbicide and antibiotic 4-formylaminooxyvinylglycine (FVG) was originally isolated from several rhizosphere-associated strains of Pseudomonas fluorescens. Biosynthesis of FVG is dependent on the gvg biosynthetic gene cluster in P. fluorescens. In this investigation, we used comparative genomics to identify strains with the genetic potential to produce FVG due to presence of a gvg gene cluster. These strains primarily belong to two groups of Pseudomonas, P. fluorescens and P. syringae, however, a few strains with the gvg cluster were found outside of Pseudomonas. Mass spectrometry confirmed that all tested strains of the P. fluorescens species group produced FVG. However, P. syringae strains did not produce FVG under standard conditions. Several lines of evidence regarding the transmission of the gvg cluster including a robust phylogenetic analysis suggest that it was introduced multiple times through horizontal gene transfer within the Pseudomonas lineage as well as in select lineages of Thiomonas, Burkholderia and Pantoea. Together, these data broaden our understanding of the evolution and diversity of FVG biosynthesis. In the course of this investigation, additional gene clusters containing only a subset of the genes required to produce FVG were identified in a broad range of bacteria, including many non-pseudomonads.

scholarly journals Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuanyuan Shi ◽  
Renjie Gu ◽  
Yihong Li ◽  
Xinwei Wang ◽  
Weicong Ren ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Consensus Sequence ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Binding Motif ◽  
Biosynthetic Gene ◽  
Structural Genes ◽  
Biosynthetic Gene Clusters ◽  
Molecular Networking ◽  
Overexpression Strain

Abstract Background Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC–MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. Results The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. Conclusions A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the “cryptic” herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

scholarly journals Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

2021 ◽  
Author(s):  
Valentin Waschulin ◽  
Chiara Borsetto ◽  
Robert James ◽  
Kevin K. Newsham ◽  
Stefano Donadio ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Full Length ◽  
Metagenomic Sequencing ◽  
Short Read ◽  
Short Read Sequencing ◽  
Rich Diversity ◽  
Long Read ◽  
The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

scholarly journals Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Emmanuel Matabaro ◽  
Hannelore Kaspar ◽  
Paul Dahlin ◽  
Daniel L. V. Bader ◽  
Claudia E. Murar ◽  
...  
Keyword(s):  
Natural Products ◽  
Lentinula Edodes ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Precursor Protein ◽  
Fungal Genome ◽  
Homologous Gene ◽  
C Terminus ◽  
Natural Variants ◽  
Recombinant Peptides

AbstractBackbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

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.

scholarly journals Identification of Putative Biosynthetic Gene Clusters for Tolyporphins in Multiple Filamentous Cyanobacteria

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 758
Author(s):  
Xiaohe Jin ◽  
Yunlong Zhang ◽  
Ran Zhang ◽  
Kathy-Uyen Nguyen ◽  
Jonathan S. Lindsey ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Filamentous Cyanobacterium ◽  
Biosynthetic Gene ◽  
Filamentous Cyanobacteria ◽  
Biosynthetic Gene Clusters ◽  
Common Component ◽  
Homology Searching ◽  
Similar Gene

Tolyporphins A–R are unusual tetrapyrrole macrocycles produced by the non-axenic filamentous cyanobacterium HT-58-2. A putative biosynthetic gene cluster for biosynthesis of tolyporphins (here termed BGC-1) was previously identified in the genome of HT-58-2. Here, homology searching of BGC-1 in HT-58-2 led to identification of similar BGCs in seven other filamentous cyanobacteria, including strains Nostoc sp. 106C, Nostoc sp. RF31YmG, Nostoc sp. FACHB-892, Brasilonema octagenarum UFV-OR1, Brasilonema octagenarum UFV-E1, Brasilonema sennae CENA114 and Oculatella sp. LEGE 06141, suggesting their potential for tolyporphins production. A similar gene cluster (BGC-2) also was identified unexpectedly in HT-58-2. Tolyporphins BGCs were not identified in unicellular cyanobacteria. Phylogenetic analysis based on 16S rRNA and a common component of the BGCs, TolD, points to a close evolutionary history between each strain and their respective tolyporphins BGC. Though identified with putative tolyporphins BGCs, examination of pigments extracted from three cyanobacteria has not revealed the presence of tolyporphins. Overall, the identification of BGCs and potential producers of tolyporphins presents a collection of candidate cyanobacteria for genetic and biochemical analysis pertaining to these unusual tetrapyrrole macrocycles.

scholarly journals The Tripod for Bacterial Natural Product Discovery: Genome Mining, Silent Pathway Induction, and Mass Spectrometry-Based Molecular Networking

mSystems ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Daniela B. B. Trivella ◽  
Rafael de Felicio
Keyword(s):  
Mass Spectrometry ◽  
Natural Products ◽  
Natural Product ◽  
Biosynthetic Pathway ◽  
Genome Mining ◽  
Chemical Compounds ◽  
Gene Clusters ◽  
Tandem Mass ◽  
Molecular Networking ◽  
Natural Product Discovery

ABSTRACT Natural products are the richest source of chemical compounds for drug discovery. Particularly, bacterial secondary metabolites are in the spotlight due to advances in genome sequencing and mining, as well as for the potential of biosynthetic pathway manipulation to awake silent (cryptic) gene clusters under laboratory cultivation. Further progress in compound detection, such as the development of the tandem mass spectrometry (MS/MS) molecular networking approach, has contributed to the discovery of novel bacterial natural products. The latter can be applied directly to bacterial crude extracts for identifying and dereplicating known compounds, therefore assisting the prioritization of extracts containing novel natural products, for example. In our opinion, these three approaches—genome mining, silent pathway induction, and MS-based molecular networking—compose the tripod for modern bacterial natural product discovery and will be discussed in this perspective.

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