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Tuesday, June 1, 2021

Genome sequencing of microbial genomes using Single Molecule Real-time sequencing (SMRT) technology.

In the last year, high-throughput sequencing technologies have progressed from proof-of-concept to production quality. Although each technology is able to produce vast quantities of sequence information, in every case the underlying chemistry limits reads to very short lengths. We present a examining de novo assembly comparison with bacterial genome assembly varying genome size (from 3.1Mb to 7.6Mb) and different G+C contents (from 43% to 71%), respectively. We analyzed Solexa reads, 454 reads and Pacbio RS reads from Streptomyces sp. (Genome size, 7.6 Mb; G+C content, 71%), Psychrobacter sp. (Genome size, 3.5 Mb; G+C content, 43%), Salinibacterium sp. (Genome size, 3.1…

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Tuesday, June 1, 2021

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence,…

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Friday, April 9, 2021

Creating Core Demand with HiFi Sequencing

In this video, Dave Miller from PacBio and Alvaro Hernandez PhD from the University of Illinois Urbana- Champaign discuss how to create Core Lab demand using PacBio highly accurate long-read, or HiFi, sequencing.

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Friday, April 9, 2021

The Long and Short of Sequencing – Why HiFi Reads are the Future 

PacBio Sequencing and software enable the generation of highly accurate (>99.9%) long reads. HiFi reads are accurate, essential, affordable, and can be used across a range of applications, including detection of all variant types, from single nucleotides to structural variants. PacBio’s end-to-end solutions feature library preparation paired with push-button analysis to support numerous workflows so you can run projects quickly and easily.

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Tuesday, April 21, 2020

The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor.

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have…

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Tuesday, April 21, 2020

Genome-Guided Discovery of Pretilactam from Actinosynnema pretiosum ATCC 31565.

Actinosynnema is a small but well-known genus of actinomycetes for production of ansamitocin, the payload component of antibody-drug conjugates against cancers. However, the secondary metabolite production profile of Actinosynnema pretiosum ATCC 31565, the most famous producer of ansamitocin, has never been fully explored. Our antiSMASH analysis of the genomic DNA of Actinosynnema pretiosum ATCC 31565 revealed a NRPS-PKS gene cluster for polyene macrolactam. The gene cluster is very similar to gene clusters for mirilactam and salinilactam, two 26-membered polyene macrolactams from Actinosynnema mirum and Salinispora tropica, respectively. Guided by this bioinformatics prediction, we characterized a novel 26-membered polyene macrolactam from…

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Tuesday, April 21, 2020

Heterologous Expression of Ilicicolin H Biosynthetic Gene Cluster and Production of a New Potent Antifungal Reagent, Ilicicolin J.

Ilicicolin H is a broad-spectrum antifungal agent targeting mitochondrial cytochrome bc1 reductase. Unfortunately, ilicicolin H shows reduced activities in vivo. Here, we report our effort on the identification of ilicicolin H biosynthetic gene cluster (BGC) by genomic sequencing a producing strain, Neonectria sp. DH2, and its heterologous production in Aspergillus nidulans. In addition, a shunt product with similar antifungal activities, ilicicolin J, was uncovered. This effort would provide a base for future combinatorial biosynthesis of ilicicolin H analogues. Bioinformatics analysis suggests that the backbone of ilicicolin H is assembled by a polyketide-nonribosomal peptide synthethase (IliA), and then offloaded with a…

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Tuesday, April 21, 2020

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting…

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Tuesday, April 21, 2020

Characterization and Complete Genome Analysis of the Carbazomycin B-Producing Strain Streptomyces luteoverticillatus SZJ61.

Members of marine Actinobacteria have been highly regarded as potentially important sources of antimicrobial compounds. Here, we isolated a strain of Actinobacteria, SZJ61, and showed that it inhibits the in vitro growth of fungi pathogenic to plants. This new isolate was identified as Streptomyces luteoverticillatus by morphological, biochemical and genetic analyses. Antifungal compounds were isolated from S. luteoverticillatus strain SZJ61 and characterized as carbazomycin B by nuclear magnetic resonance spectra. We then sequenced the genome of the S. luteoverticillatus SZJ61 strain, which consists of only one 7,367,863 bp linear chromosome that has a G+C content of 72.05%. Thirty-five putative biosynthetic gene…

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Tuesday, April 21, 2020

Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066.

Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D (1-4). Their connectivity was determined by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochemistry of each stereocluster in these compounds was established using NOE analysis, the universal database method and J-based configuration analysis, further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochemical outcome…

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Tuesday, April 21, 2020

Natural product drug discovery in the genomic era: realities, conjectures, misconceptions, and opportunities.

Natural product discovery from microorganisms provided important sources for antibiotics, anti-cancer agents, immune-modulators, anthelminthic agents, and insecticides during a span of 50 years starting in the 1940s, then became less productive because of rediscovery issues, low throughput, and lack of relevant new technologies to unveil less abundant or not easily detected drug-like natural products. In the early 2000s, it was observed from genome sequencing that Streptomyces species encode about ten times as many secondary metabolites as predicted from known secondary metabolomes. This gave rise to a new discovery approach-microbial genome mining. As the cost of genome sequencing dropped, the numbers of…

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Tuesday, April 21, 2020

Cytotoxic and Antibacterial Cervinomycins B1-4 from a Streptomyces Species.

AntiSMASH analysis of genome DNA of Streptomyces CPCC 204980, a soil isolate with potent antibacterial activity, revealed a gene cluster for polycyclic xanthones. A subsequent chemical study confirmed that the microorganism produced polycyclic xanthone cervinomycin A2 (1) and the new congeners cervinomycins B1-4 (2-5). The structures of 1-5 were determined by comprehensive analyses of MS and NMR data, which indicated that 2-5 featured a common dihydro-D ring in the polycyclic xanthone core moiety of their molecules. 2-5 are toxic to human cancer cells and active against Gram-positive bacteria.

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Tuesday, April 21, 2020

Isarubrolones Containing a Pyridooxazinium Unit from Streptomyces as Autophagy Activators.

Isarubrolones are bioactive polycyclic tropoloalkaloids from Streptomyces. Three new isarubrolones (2-4), together with the known isarubrolone C (1) and isatropolones A (5) and C (6, 3( R)-hydroxyisatropolone A), were identified from Streptomyces sp. CPCC 204095. The structures of these compounds were determined using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and ECD. Compounds 3 and 4 feature a pyridooxazinium unit, which is rarely seen in natural products. Compound 6 could conjugate with amino acids or amines to expand the structural diversity of isarubrolones with a pentacyclic or hexacyclic core. Importantly, 1 and 3-6 were found to induce…

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Tuesday, April 21, 2020

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools…

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