Expansion of gamma-butyrolactone signaling molecule biosynthesis to phosphotriester natural products

AbstractBacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their anti-malarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the saliniphostins and newly identified A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed amongst many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

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

Journal of Bacteriology ◽

10.1128/jb.00346-18 ◽

2018 ◽

Vol 200 (21) ◽

Cited By ~ 8

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.

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In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

mBio ◽

10.1128/mbio.01714-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 28

Author(s):

Yunkun Liu ◽

Weixin Tao ◽

Shishi Wen ◽

Zhengyuan Li ◽

Anna Yang ◽

...

Keyword(s):

Dna Polymerase ◽

Gene Clusters ◽

Specific Gene ◽

Dna Fragments ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Highly Efficient ◽

Dna Editing

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. IMPORTANCE Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

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Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

10.1101/365031 ◽

2018 ◽

Author(s):

Kwo-Kwang Abraham Wang ◽

Tai L. Ng ◽

Peng Wang ◽

Zedu Huang ◽

Emily P. Balskus ◽

...

Keyword(s):

Natural Products ◽

Nitrous Acid ◽

Bond Formation ◽

Gene Clusters ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Structural Differences ◽

Related Compounds ◽

Made In

AbstractFosfazinomycin and kinamycin are natural products that contain nitrogen-nitrogen (N-N) bonds but that are otherwise structurally unrelated. Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation. In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid. Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways. Therefore, unlike other pathways to NN bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products.

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Complex evolutionary dynamics govern the diversity and distribution of biosynthetic gene clusters and their encoded specialized metabolites

10.1101/2020.12.19.423547 ◽

2020 ◽

Author(s):

Alexander B. Chase ◽

Douglas Sweeney ◽

Mitchell N. Muskat ◽

Dulce Guillén-Matus ◽

Paul R. Jensen

Keyword(s):

Evolutionary Dynamics ◽

Gene Clusters ◽

Ecological Interactions ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Evolutionary Processes ◽

Specialized Metabolites ◽

Marine Actinomycete ◽

Species Specific ◽

Gain Loss

ABSTRACTWhile specialized metabolites are thought to mediate ecological interactions, the evolutionary processes driving their distributions, particularly among closely related lineages, remain poorly understood. Here, we examine the evolutionary dynamics governing the diversity and distribution of biosynthetic gene clusters (BGCs) in 118 strains across nine described species within the marine actinomycete genus Salinispora. While previous evidence indicated that horizontal gene transfer largely contributed to BGC diversity, we find that a majority of BGCs in Salinispora genomes are maintained by processes of vertical descent. In particular, we identified species-specific signatures that were associated with both BGC distributions and the production of their encoded specialized metabolites. By analyzing nine experimentally characterized BGCs that range in conservation from species to genus specific, we find that the distribution of BGCs among Salinispora species is maintained by selection, while BGC diversification is constrained by recombination among closely related strains and strengthened by gain/loss events between species. Notably, the evolutionary processes driving BGC diversification had direct consequences for compound production, elucidating the mechanisms that lead to chemical diversification. These results support the concept that specialized metabolites, and their cognate BGCs, represent functional traits associated with ecological differentiation among Salinispora species.GRAPHICAL ABSTRACT

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A widespread family of bacterial gene clusters produces ClpP inhibitors

10.21203/rs.3.rs-464484/v1 ◽

2021 ◽

Author(s):

Gerry Wright ◽

Elizabeth Culp ◽

David Sychantha ◽

Christian Hobson ◽

Andrew Pawlowski ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Genome Mining ◽

Antibiotic Resistance Genes ◽

Gene Clusters ◽

Biosynthetic Gene Clusters ◽

Bacterial Gene ◽

Clinical Potential ◽

Novel Scaffold

Abstract Intracellular proteolytic complexes play an essential role in modeling the proteome in both bacteria and eukaryotes. ClpP is the protease subunit of one such highly conserved proteolytic complex that, despite its potential, remains unexploited as a drug target. Here we describe a target-directed genome mining strategy to identify ClpP targeting compounds from the bacterial order Actinomycetales. By searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes, we identify a family of ClpP-associated clusters that are widespread across phyla, including environmental and pathogenic bacteria. While numerous bacterial pyrrolizidine alkaloids produced by these gene clusters are known, their connection to ClpP has never been made. We show that these previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya DSM 46488, we use heterologous expression to purify the relevant ClpP inhibitor, which we name clipibicyclene. We show in vitro and in vivo that clipibicyclene is a potent covalent inhibitor of ClpP and that cluster-associated ClpPs provide resistance. ClpP inhibition results in antibacterial activity against actinobacteria, including Mycobacterium smegmatis, and inhibition of virulence factor production by Staphylococcus aureus. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene’s discovery deconvolutes the actual function of a family of natural products widespread in nature. It provides a novel scaffold for therapeutic ClpP inhibitor development, making these findings significant from the perspective of their discovery and their clinical potential.

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Structural evolution of a DNA repair self-resistance mechanism targeting genotoxic secondary metabolites

Nature Communications ◽

10.1038/s41467-021-27284-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Elwood A. Mullins ◽

Jonathan Dorival ◽

Gong-Li Tang ◽

Dale L. Boger ◽

Brandt F. Eichman

Keyword(s):

Natural Products ◽

Selective Pressure ◽

Alkylating Agents ◽

Resistance Mechanism ◽

Structural Evolution ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Structural Basis

AbstractMicrobes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity—not enhanced substrate recognition—is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065.

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An interpreted atlas of biosynthetic gene clusters from 1,000 fungal genomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020230118 ◽

2021 ◽

Vol 118 (19) ◽

pp. e2020230118

Author(s):

Matthew T. Robey ◽

Lindsay K. Caesar ◽

Milton T. Drott ◽

Nancy P. Keller ◽

Neil L. Kelleher

Keyword(s):

Natural Products ◽

Chemical Space ◽

Genome Mining ◽

Fold Increase ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Automated Annotation ◽

Fungal Genomes ◽

Species Specific

Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1,000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here, we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1,037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of fungi, organizing 15,213 fungal compounds into 2,945 molecular families (MFs). The taxonomic landscape of fungal GCFs is largely species specific, though select families such as the equisetin GCF are present across vast phylogenetic distances with parallel diversifications in the GCF and MF. We compare these fungal datasets with a set of 5,453 bacterial genomes and their BGCs and 9,382 bacterial compounds, revealing dramatic differences between bacterial and fungal biosynthetic logic and chemical space. These genomics and cheminformatics analyses reveal the large extent to which fungal and bacterial sources represent distinct compound reservoirs. With a >10-fold increase in the number of interpreted strains and annotated BGCs, this work better regularizes the biosynthetic potential of fungi for rational compound discovery.

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

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

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.

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Natural Anti-inflammatory compounds as Drug candidates in Alzheimer’s disease

Current Medicinal Chemistry ◽

10.2174/0929867327666200730213215 ◽

2020 ◽

Vol 27 ◽

Author(s):

Reyaz Hassan Mir ◽

Abdul Jalil Shah ◽

Roohi Mohi-ud-din ◽

Faheem Hyder Potoo ◽

Mohd. Akbar Dar ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Natural Products ◽

Protective Action ◽

New Drugs ◽

Huperzine A ◽

Brain Disorder ◽

Anti Inflammatory

: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder characterized by memory impairment, dementia, oxidative stress in elderly people. Currently, only a few drugs are available in the market with various adverse effects. So to develop new drugs with protective action against the disease, research is turning to the identification of plant products as a remedy. Natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Phytochemicals including Curcumin, Resveratrol, Quercetin, Huperzine-A, Rosmarinic acid, genistein, obovatol, and Oxyresvertarol were reported molecules for the treatment of AD. Several alkaloids such as galantamine, oridonin, glaucocalyxin B, tetrandrine, berberine, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in-vivo. In conclusion, natural products from plants represent interesting candidates for the treatment of AD. This review highlights the potential of specific compounds from natural products along with their synthetic derivatives to counteract AD in the CNS.

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Potential for Treatment of Neurodegenerative Diseases with Natural Products or Synthetic Compounds that Stabilize Microtubules

Current Pharmaceutical Design ◽

10.2174/1381612826666200621171302 ◽

2020 ◽

Vol 26 (35) ◽

pp. 4362-4372

Author(s):

John H. Miller ◽

Viswanath Das

Keyword(s):

Natural Products ◽

Neurodegenerative Diseases ◽

In Vivo Models ◽

Synthetic Compounds ◽

Stabilizing Agents ◽

Animal Models Of Disease ◽

Model Studies ◽

Models Of Disease

No effective therapeutics to treat neurodegenerative diseases exist, despite significant attempts to find drugs that can reduce or rescue the debilitating symptoms of tauopathies such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, or Pick’s disease. A number of in vitro and in vivo models exist for studying neurodegenerative diseases, including cell models employing induced-pluripotent stem cells, cerebral organoids, and animal models of disease. Recent research has focused on microtubulestabilizing agents, either natural products or synthetic compounds that can prevent the axonal destruction caused by tau protein pathologies. Although promising results have come from animal model studies using brainpenetrant natural product microtubule-stabilizing agents, such as paclitaxel analogs that can access the brain, epothilones B and D, and other synthetic compounds such as davunetide or the triazolopyrimidines, early clinical trials in humans have been disappointing. This review aims to summarize the research that has been carried out in this area and discuss the potential for the future development of an effective microtubule stabilizing drug to treat neurodegenerative disease.

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