scholarly journals Discovery and Heterologous Production of New Cyclic Depsibosamycins

2021 ◽  
Vol 9 (7) ◽  
pp. 1396
Author(s):  
Marc Stierhof ◽  
Maksym Myronovskyi ◽  
Josef Zapp ◽  
Andriy Luzhetskyy
Keyword(s):  
Cyclic Peptides ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Side Chain ◽  
Biological Functions ◽  
Biosynthetic Gene Clusters ◽  
Streptomyces Species ◽  
Peptide Synthetases ◽  
Major Interest ◽  
In Silico Identification

Streptomyces are producers of valuable secondary metabolites with unique scaffolds that perform a plethora of biological functions. Nonribosomal peptides are of special interest due to their variety and complexity. They are synthesized by nonribosomal peptide synthetases, large biosynthetic machineries that are encoded in the genome of many Streptomyces species. The identification of new peptides and the corresponding biosynthetic gene clusters is of major interest since knowledge can be used to facilitate combinatorial biosynthesis and chemical semisynthesis of natural products. The recently discovered bosamycins are linear octapeptides with an interesting 5-OMe tyrosine moiety and various modifications at the N-terminus. In this study, the new cyclic depsibosamycins B, C, and D from Streptomyces aurantiacus LU19075 were discovered. In comparison to the linear bosamycins B, C, and D, which were also produced by the strain, the cyclic depsibosamycins showed a side-chain-to-tail lactonization of serine and glycine, leading to a ring of four amino acids. In silico identification and heterologous expression of the depsibosamycin (dbm) gene cluster indicated that the cyclic peptides, rather than the linear derivatives, are the main products of the cluster.

scholarly journals Inter-Modular Linkers play a crucial role in governing the biosynthesis of non-ribosomal peptides

2019 ◽  
Vol 35 (19) ◽  
pp. 3584-3591 ◽  
Author(s):  
Sherif Farag ◽  
Rachel M Bleich ◽  
Elizabeth A Shank ◽  
Olexandr Isayev ◽  
Albert A Bowers ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Clinical Applications ◽  
Combinatorial Biosynthesis ◽  
Supplementary Information ◽  
Biosynthetic Gene ◽  
Small Peptides ◽  
Bacterial Genomes ◽  
Biosynthetic Gene Clusters ◽  
Peptide Synthetases ◽  
Anti Cancer

Abstract Motivation Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic machines that catalyze the ribosome-independent production of structurally complex small peptides, many of which have important clinical applications as antibiotics, antifungals and anti-cancer agents. Several groups have tried to expand natural product diversity by intermixing different NRPS modules to create synthetic peptides. This approach has not been as successful as anticipated, suggesting that these modules are not fully interchangeable. Results We explored whether Inter-Modular Linkers (IMLs) impact the ability of NRPS modules to communicate during the synthesis of NRPs. We developed a parser to extract 39 804 IMLs from both well annotated and putative NRPS biosynthetic gene clusters from 39 232 bacterial genomes and established the first IMLs database. We analyzed these IMLs and identified a striking relationship between IMLs and the amino acid substrates of their adjacent modules. More than 92% of the identified IMLs connect modules that activate a particular pair of substrates, suggesting that significant specificity is embedded within these sequences. We therefore propose that incorporating the correct IML is critical when attempting combinatorial biosynthesis of novel NRPS. Availability and implementation The IMLs database as well as the NRPS-Parser have been made available on the web at https://nrps-linker.unc.edu. The entire source code of the project is hosted in GitHub repository (https://github.com/SWFarag/nrps-linker). Supplementary information Supplementary data are available at Bioinformatics online.

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.

Future directions for the discovery of antibiotics from actinomycete bacteria

2017 ◽  
Vol 1 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Rebecca Devine ◽  
Matthew I. Hutchings ◽  
Neil A. Holmes
Keyword(s):  
New Technologies ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Future Directions ◽  
Streptomyces Species ◽  
Genus Streptomyces ◽  
New Antibiotics ◽  
The Uk ◽  
Almost All ◽  
New Generation

Antimicrobial resistance (AMR) is a growing societal problem, and without new anti-infective drugs, the UK government-commissioned O'Neil report has predicted that infectious disease will claim the lives of an additional 10 million people a year worldwide by 2050. Almost all the antibiotics currently in clinical use are derived from the secondary metabolites of a group of filamentous soil bacteria called actinomycetes, most notably in the genus Streptomyces. Unfortunately, the discovery of these strains and their natural products (NPs) peaked in the 1950s and was then largely abandoned, partly due to the repeated rediscovery of known strains and compounds. Attention turned instead to rational target-based drug design, but this was largely unsuccessful and few new antibiotics have made it to clinic in the last 60 years. In the early 2000s, however, genome sequencing of the first Streptomyces species reinvigorated interest in NP discovery because it revealed the presence of numerous cryptic NP biosynthetic gene clusters that are not expressed in the laboratory. Here, we describe how the use of new technologies, including improved culture-dependent and -independent techniques, combined with searching underexplored environments, promises to identify a new generation of NP antibiotics from actinomycete bacteria.

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

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 Secondary metabolite biosynthetic diversity in Arctic Ocean metagenomes

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Adriana Rego ◽  
Antonio Fernandez-Guerra ◽  
Pedro Duarte ◽  
Philipp Assmy ◽  
Pedro N. Leão ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Arctic Ocean ◽  
High Throughput Sequencing ◽  
Large Fraction ◽  
Environmental Dna ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Bacterial Isolation ◽  
Biosynthetic Genes ◽  
Peptide Synthetases

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are mega enzymes responsible for the biosynthesis of a large fraction of natural products (NPs). Molecular markers for biosynthetic genes, such as the ketosynthase (KS) domain of PKSs, have been used to assess the diversity and distribution of biosynthetic genes in complex microbial communities. More recently, metagenomic studies have complemented and enhanced this approach by allowing the recovery of complete biosynthetic gene clusters (BGCs) from environmental DNA. In this study, the distribution and diversity of biosynthetic genes and clusters from Arctic Ocean samples (NICE-2015 expedition), was assessed using PCR-based strategies coupled with high-throughput sequencing and metagenomic analysis. In total, 149 KS domain OTU sequences were recovered, 36 % of which could not be assigned to any known BGC. In addition, 74 bacterial metagenome-assembled genomes were recovered, from which 179 BGCs were extracted. A network analysis identified potential new NP families, including non-ribosomal peptides and polyketides. Complete or near-complete BGCs were recovered, which will enable future heterologous expression efforts to uncover the respective NPs. Our study represents the first report of biosynthetic diversity assessed for Arctic Ocean metagenomes and highlights the potential of Arctic Ocean planktonic microbiomes for the discovery of novel secondary metabolites. The strategy employed in this study will enable future bioprospection, by identifying promising samples for bacterial isolation efforts, while providing also full-length BGCs for heterologous expression.

scholarly journals Whole genome sequence of Streptomyces humi strain MUSC 119T isolated from intertidal soil

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Hooi-Leng Ser ◽  
Wen-Si Tan ◽  
Wai-Fong Yin ◽  
Kok-Gan Chan ◽  
Nurul Syakima Ab Mutalib ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Biosynthetic Gene Clusters ◽  
Streptomyces Species ◽  
Genus Streptomyces ◽  
The Past ◽  
Mangrove Soil ◽  
Natural Product Research ◽  
Biochemical Assays

Over the past few decades, microorganisms have made major contribution in natural product research, particularly those from the genus Streptomyces. Streptomyces humi MUSC 119T was previously isolated as novel streptomycete from mangrove soil in Malaysia. During the screening programme for bioactive strains, this strain was discovered to possess antioxidant activity – scavenging and reducing accumulation of free radicals in biochemical assays. Consequently, whole genome sequencing was performed to evaluate genomic potential of the strain. Based on our analysis, the genome size of MUSC 119T is described to be 10.01 Mbps with G + C content of 71.80%. Based on antiSMASH analysis, the strain possess great genomic potential, having nine biosynthetic gene clusters displaying high similarities to known gene clusters. These findings indicates that mangrove Streptomyces species like MUSC 119T may potentially play an important role in drug development process, while the availability of its whole genome sequences allows further manipulation to isolate and identify compound of interest.

scholarly journals Microcystin Biosynthesis in Planktothrix: Genes, Evolution, and Manipulation

2003 ◽  
Vol 185 (2) ◽  
pp. 564-572 ◽  
Author(s):  
Guntram Christiansen ◽  
Jutta Fastner ◽  
Marcel Erhard ◽  
Thomas Börner ◽  
Elke Dittmann
Keyword(s):  
Protein Phosphatases ◽  
Large Family ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Filamentous Cyanobacterium ◽  
Unicellular Cyanobacterium ◽  
Peptide Synthetases ◽  
Microcystin Synthetase ◽  
Targeted Mutation ◽  
Gene Coding

ABSTRACT Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.

scholarly journals Ancient Bacterial Class Alphaproteobacteria Cytochrome P450 Monooxygenases Can Be Found in Other Bacterial Species

2021 ◽  
Vol 22 (11) ◽  
pp. 5542
Author(s):  
Nomfundo Nzuza ◽  
Tiara Padayachee ◽  
Puleng Rosinah Syed ◽  
Justyna Dorota Kryś ◽  
Wanping Chen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Side Chain ◽  
Cytochrome P450 Monooxygenases ◽  
Biosynthetic Gene Clusters ◽  
Genome Data ◽  
P450 Monooxygenases ◽  
Large Numbers ◽  
Cyanobacterial Species

Cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins, are well-known players in the generation of chemicals valuable to humans and as a drug target against pathogens. Understanding the evolution of P450s in a bacterial population is gaining momentum. In this study, we report comprehensive analysis of P450s in the ancient group of the bacterial class Alphaproteobacteria. Genome data mining and annotation of P450s in 599 alphaproteobacterial species belonging to 164 genera revealed the presence of P450s in only 241 species belonging to 82 genera that are grouped into 143 P450 families and 214 P450 subfamilies, including 77 new P450 families. Alphaproteobacterial species have the highest average number of P450s compared to Firmicutes species and cyanobacterial species. The lowest percentage of alphaproteobacterial species P450s (2.4%) was found to be part of secondary metabolite biosynthetic gene clusters (BGCs), compared other bacterial species, indicating that during evolution large numbers of P450s became part of BGCs in other bacterial species. Our study identified that some of the P450 families found in alphaproteobacterial species were passed to other bacterial species. This is the first study to report on the identification of CYP125 P450, cholesterol and cholest-4-en-3-one hydroxylase in alphaproteobacterial species (Phenylobacterium zucineum) and to predict cholesterol side-chain oxidation capability (based on homolog proteins) by P. zucineum.

scholarly journals BioCAT: PSSM-based algorithm to search biosynthetic gene clusters producing nonribosomal peptides with known structure

2021 ◽  
Author(s):  
Dmitry N Konanov ◽  
Danil V Krivonos ◽  
Vladislav V Babenko ◽  
Elena N Ilina
Keyword(s):  
Chemical Structure ◽  
Gene Clusters ◽  
Nonribosomal Peptides ◽  
Biosynthetic Gene Clusters ◽  
High Priority Task ◽  
Peptide Synthetases ◽  
Priority Task ◽  
Modern Biotechnology ◽  
Bacteria And Fungi ◽  
Final Alignment

Motivation: Nonribosomal peptides (NRPs) are a class of secondary metabolites synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSs) and mainly produced by bacteria and fungi. It has been shown that NRPs have a huge structural and functional diversity including antimicrobial activity, therefore, they are of increasing interest for modern biotechnology. Methods such as NMR and LC-MS/MS allow to determine NRP structure precisely, but it is often not a trivial task to find natural producers of them. Today, searches are usually performed manually, mostly with tools such as antiSMASH or Prism. However, there are cases when potential producers should be found among hundreds of strains, for instance, when analyzing metagenomes data. Thus, the development of automated approaches is a high-priority task for further NRP research. Results: We developed BioCAT, a two-side approach to find biosynthesys gene clusters (BGCs) which may produce a given NRP when the structure of interesting NRP has already been found. Formally, the BioCAT unites the antiSMASH software and the rBAN retrosynthesis tool but some improvements were added to both gene cluster and NRP chemical structure analyses. The main feature of the method is PSSM usage to store specificities of NRPS modules, which has increased the alignment quality in comparison with more strict approaches developed earlier. An ensemble model was implemented to calculate the final alignment score. We tested the method on a manually curated NRP producers database and compared it with a competing tool called GARLIC. Finally, we showed the method applicability on a several external examples.

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