Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468

ABSTRACT A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif. This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms. IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

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Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin

Applied and Environmental Microbiology ◽

10.1128/aem.01752-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 5

Author(s):

Juan Pablo Gomez-Escribano ◽

Jean Franco Castro ◽

Valeria Razmilic ◽

Scott A. Jarmusch ◽

Gerhard Saalbach ◽

...

Keyword(s):

Natural Products ◽

Heterologous Expression ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Growth Media ◽

Content Type ◽

Lasso Peptide ◽

Lasso Peptides ◽

New Family

ABSTRACT Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides. IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

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Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

Applied and Environmental Microbiology ◽

10.1128/aem.00038-11 ◽

2011 ◽

Vol 77 (11) ◽

pp. 3617-3625 ◽

Cited By ~ 66

Author(s):

Daniel W. Udwary ◽

Erin A. Gontang ◽

Adam C. Jones ◽

Carla S. Jones ◽

Andrew W. Schultz ◽

...

Keyword(s):

Natural Products ◽

Cyclic Peptides ◽

Intact Cell ◽

Gene Clusters ◽

Mass Range ◽

Bioinformatic Analysis ◽

Comparative Genomic ◽

Ionization Mass ◽

Content Type ◽

Species Specific

ABSTRACTBacteria of the genusFrankiaare mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genusFrankiahas largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences ofFrankiastrains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from otherFrankiastrains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis ofFrankiasp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed inFrankiasp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in theFrankiagenus.

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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 Anabaena 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 Anabaena7120 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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WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

Applied and Environmental Microbiology ◽

10.1128/aem.01849-14 ◽

2014 ◽

Vol 80 (22) ◽

pp. 6879-6887 ◽

Cited By ~ 22

Author(s):

Pin Yu ◽

Shui-Ping Liu ◽

Qing-Ting Bu ◽

Zhen-Xing Zhou ◽

Zhen-Hong Zhu ◽

...

Keyword(s):

Morphological Differentiation ◽

Binding Activity ◽

Transcriptional Analysis ◽

Antibiotic Biosynthesis ◽

Content Type ◽

Real Time Quantitative Pcr ◽

Dna Binding Activity ◽

In The Wild ◽

Natamycin Production ◽

Streptomyces Chattanoogensis

ABSTRACTDetailed mechanisms ofWhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, aStreptomyces chattanoogensisL10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression ofwblAchwas shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAchhas specific DNA-binding activity for the promoter region ofwblAch. Gene disruption and genetic complementation revealed that WblAchacts in a positive manner to regulate natamycin production. WhenwblAchwas overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels ofwhigenes (includingwhiA,whiB,whiH, andwhiI) were severely depressed in the ΔwblAchmutant, suggesting that WblAchplays a part in morphological differentiation by influencing the expression of thewhigenes.

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Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03512-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6185-6196 ◽

Cited By ~ 51

Author(s):

Marius Spohn ◽

Norbert Kirchner ◽

Andreas Kulik ◽

Angelika Jochim ◽

Felix Wolf ◽

...

Keyword(s):

Mass Spectrometry ◽

Gene Cluster ◽

High Performance ◽

Pathogenic Bacteria ◽

Genome Mining ◽

Gene Clusters ◽

Von Willebrand Disease ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

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Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

Applied and Environmental Microbiology ◽

10.1128/aem.01015-17 ◽

2017 ◽

Vol 83 (17) ◽

Cited By ~ 24

Author(s):

Domonique A. Carson ◽

Herman W. Barkema ◽

Sohail Naushad ◽

Jeroen De Buck

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Genome Mining ◽

Bovine Milk ◽

Gene Clusters ◽

Antibiotic Use ◽

Content Type ◽

Treatment And Prevention ◽

Whole Genomes

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

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

mSystems ◽

10.1128/msystems.00160-17 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 14

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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Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

10.26686/wgtn.17147699.v1 ◽

2021 ◽

Author(s):

◽

Luke Stevenson

Keyword(s):

Natural Products ◽

New Zealand ◽

Heterologous Expression ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Coli Strain ◽

Functional Screening ◽

Functional Difference ◽

Biosynthetic Pathways ◽

New Members

Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.

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Characterization and analysis of an industrial strain of Streptomyces bingchenggensis by genome sequencing and gene microarray

Genome ◽

10.1139/gen-2013-0098 ◽

2013 ◽

Vol 56 (11) ◽

pp. 677-689 ◽

Cited By ~ 7

Author(s):

Xiang-Jing Wang ◽

Bo Zhang ◽

Yi-Jun Yan ◽

Jing An ◽

Ji Zhang ◽

...

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Crop Protection ◽

Gc Content ◽

Gene Clusters ◽

High Expression ◽

Expression Level ◽

Transcriptional Analysis ◽

Streptomyces Bingchenggensis ◽

High Expression Level

Streptomyces bingchenggensis is a soil bacterium that produces milbemycins, a family of macrolide antibiotics that are commercially important in crop protection and veterinary medicine. In addition, S. bingchenggensis produces many other natural products including the polyether nanchangmycin and novel cyclic pentapeptides. To identify the gene clusters involved in the biosynthesis of these compounds, and better clarify the biochemical pathways of these gene clusters, the whole genome of S. bingchenggensis was sequenced, and the transcriptome profile was subsequently investigated by microarray. In comparison with other sequenced genomes in Streptomyces, S. bingchenggensis has the largest linear chromosome consisting of 11 936 683 base pairs (bp), with an average GC content of 70.8%. The 10 023 predicted protein-coding sequences include at least 47 gene clusters correlated with the biosynthesis of known or predicted secondary metabolites. Transcriptional analysis demonstrated an extremely high expression level of the milbemycin gene cluster during the entire growth period and a moderately high expression level of the nanchangmycin gene cluster during the initial hours that subsequently decreased. However, other gene clusters appear to be silent. The genome-wide analysis of the secondary metabolite gene clusters in S. bingchenggensis, coupled with transcriptional analysis, will facilitate the rational development of high milbemycins-producing strains as well as the discovery of new natural products.

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Ascomycete Aspergillus oryzae Is an Efficient Expression Host for Production of Basidiomycete Terpenes by Using Genomic DNA Sequences

Applied and Environmental Microbiology ◽

10.1128/aem.00409-19 ◽

2019 ◽

Vol 85 (15) ◽

Cited By ~ 8

Author(s):

Shota Nagamine ◽

Chengwei Liu ◽

Jumpei Nishishita ◽

Takuto Kozaki ◽

Kaho Sogahata ◽

...

Keyword(s):

Natural Products ◽

Aspergillus Oryzae ◽

Dna Sequences ◽

Genomic Dna ◽

Genome Mining ◽

Biologically Active ◽

Biosynthetic Genes ◽

Content Type ◽

Content System ◽

Basidiomycete Fungi

ABSTRACT Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

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