scholarly journals Biosynthetic Potential of a Novel Antarctic Actinobacterium Marisediminicola antarctica ZS314T Revealed by Genomic Data Mining and Pigment Characterization

Marine Drugs ◽  
2019 ◽  
Vol 17 (7) ◽  
pp. 388 ◽  
Author(s):  
Li Liao ◽  
Shiyuan Su ◽  
Bin Zhao ◽  
Chengqi Fan ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Data Mining ◽  
Natural Products ◽  
Gene Cluster ◽  
Genomic Data ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
The Novel ◽  
Biosynthetic Gene ◽  
Base Pairs ◽  
Peptide Synthetase

Rare actinobacterial species are considered as potential resources of new natural products. Marisediminicola antarctica ZS314T is the only type strain of the novel actinobacterial genus Marisediminicola isolated from intertidal sediments in East Antarctica. The strain ZS314T was able to produce reddish orange pigments at low temperatures, showing characteristics of carotenoids. To understand the biosynthetic potential of this strain, the genome was completely sequenced for data mining. The complete genome had 3,352,609 base pairs (bp), much smaller than most genomes of actinomycetes. Five biosynthetic gene clusters (BGCs) were predicted in the genome, including a gene cluster responsible for the biosynthesis of C50 carotenoid, and four additional BGCs of unknown oligosaccharide, salinixanthin, alkylresorcinol derivatives, and NRPS (non-ribosomal peptide synthetase) or amino acid-derived compounds. Further experimental characterization indicated that the strain may produce C.p.450-like carotenoids, supporting the genomic data analysis. A new xanthorhodopsin gene was discovered along with the analysis of the salinixanthin biosynthetic gene cluster. Since little is known about this genus, this work improves our understanding of its biosynthetic potential and provides opportunities for further investigation of natural products and strategies for adaptation to the extreme Antarctic environment.

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

scholarly journals Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters in Actinobacteria

2017 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Metal Transport ◽  
Homologous Gene ◽  
Biosynthetic Gene ◽  
New Family ◽  
Peptide Synthetase

AbstractA putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in intro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a new family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a non-heme iron(II)-dependent oxidase homologue, and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a non-ribosomal peptide synthetase (NRPS). In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.Significance StatementMycobacterium tuberculosis is the leading causative agent of tuberculosis (TB), of which millions of deaths occur annually. A putative lipopeptide biosynthetic gene cluster has been shown to be essential for the survival of this pathogen in hosts, and homologous gene clusters have also been found in all pathogenic mycobacteria and other species of Actinobacteria. We have identified the function of these gene clusters in making a new family of isonitrile lipopeptides. The biosynthesis has several unique features, including an unprecedented mechanism for isonitrile synthesis. Our results have further suggested that these biosynthetic gene clusters play a role in metal transport, and thus have shed light on a new metal transport system that is crucial for virulence of pathogenic mycobacteria.

Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

2017 ◽  
Author(s):  
Joana Martins ◽  
Niina Leikoski ◽  
Matti Wahlsten ◽  
Joana Azevedo ◽  
Jorge Antunes ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cyclic Peptides ◽  
Biosynthetic Gene Cluster ◽  
The Novel ◽  
Tyrosine Residue ◽  
Biosynthetic Gene ◽  
Post Translational Modification ◽  
Biological Assays ◽  
Mass Spectrometric Data ◽  
Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus <i>Anabaena</i>. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (<b>1</b>), from <i>Sphaerospermopsis</i> sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (<i>acy</i>) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in <i>Escherichia</i> <i>coli</i> established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of <b>1</b> using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound <b>1</b> was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium <i>Halomonas aquamarina</i> CECT 5000.<br>

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 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 Pentaminomycins F and G, First Non-Ribosomal Peptides Containing 2-Pyridylalanine

2020 ◽  
Author(s):  
Daniel Carretero Molina ◽  
Francisco Javier Ortiz-Lopez ◽  
Jesús Martín ◽  
Ignacio González ◽  
Marina Sánchez-Hidalgo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Peptide Synthetase ◽  
Non Ribosomal Peptides

Pentaminomycins F-H, a group of three new hydroxyarginine-containing cyclic pentapeptides, were isolated from cultures of a <i>Streptomyces cacaoi</i> subsp. <i>cacaoi</i> strain along with the known pentaminomycins A-E. The structures of the new peptides were determined by a combination of mass spectrometry and NMR and Marfey's analyses. Among them, pentaminomycins F and G were shown to contain in their structures the rare amino acid 3-(2-pyridyl)-alanine. This finding represents the first reported example of non-ribosomal peptides containing this residue. The LDLLD chiral sequence found for the three compounds was in agreement with that reported for previously isolated pentaminomycins and consistent with the epimerization domains present in the putative non-robosomal peptide synthetase (NRPS) biosynthetic gene cluster.<br>

scholarly journals Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

2020 ◽  
Author(s):  
Rebecca Devine ◽  
Hannah McDonald ◽  
Zhiwei Qin ◽  
Corinne Arnold ◽  
Katie Noble ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Large Scale ◽  
Liquid Culture ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

scholarly journals mSphere of Influence: Synthetic Biology of Natural Product Biosynthesis

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Mark C. Walker
Keyword(s):  
Natural Products ◽  
Synthetic Biology ◽  
Natural Product ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Natural Product Biosynthesis ◽  
Antibiotic Compounds ◽  
Approaches To Study

ABSTRACT Mark Walker studies the biosynthesis and engineering of bacterial natural products with the long-term goal of identifying new antibiotic compounds. In this mSphere of Influence, he reflects on how “Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A” by K. Yamanaka, K. A. Reynolds, R. D. Kersten, K. S. Ryan, et al. (Proc Natl Acad Sci USA 111:1957–1962, 2014, https://doi.org/10.1073/pnas.1319584111) impacted his thinking on using synthetic biology approaches to study natural product biosynthesis.

scholarly journals Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

2019 ◽  
Vol 116 (40) ◽  
pp. 19805-19814 ◽  
Author(s):  
Zachary L. Reitz ◽  
Clifford D. Hardy ◽  
Jaewon Suk ◽  
Jean Bouvet ◽  
Alison Butler
Keyword(s):  
Predictive Power ◽  
Genome Mining ◽  
Genomic Analysis ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Peptide Synthetase ◽  
Condensation Domain

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

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