Publications by authors named "Lijiang Song"

72 Publications

Autophagy deficiency activates rDNA transcription.

Autophagy 2021 Oct 6:1-12. Epub 2021 Oct 6.

Department of Biochemistry, and Department of Thoracic Surgery of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Macroautophagy/autophagy, a highly conserved lysosome-dependent degradation pathway, has been intensively studied in regulating cell metabolism by degradation of intracellular components. In this study, we link autophagy to RNA metabolism by uncovering a regulatory role of autophagy in ribosomal RNA (rRNA) synthesis. Autophagy-deficient cells exhibit much higher precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling and promotes the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoters, which leads to an increase of rRNA transcribed from rDNA. Functionally, autophagy deficiency promotes protein synthesis, cell growth and cell proliferation, both of which are dependent on SQSTM1 accumulation. Taken together, our findings suggest that autophagy deficiency is involved in RNA metabolism by activating rDNA transcription and provide novel mechanisms for the reprogramming of cell metabolism in autophagy-related diseases including multiple types of cancers. 5-FUrd: 5-fluorouridine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A/RPA194: RNA polymerase I subunit A; POLR2A: RNA polymerase II subunit A; rDNA: ribosomal DNA; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; rRNA: ribosomal RNA; RUBCN/Rubicon: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; SUnSET: surface sensing of translation; TAX1BP1: Tax1 binding protein 1; UBTF/UBF1: upstream binding transcription factor; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild-type.
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http://dx.doi.org/10.1080/15548627.2021.1974178DOI Listing
October 2021

Synthesis of the C1-C27 Fragment of Stambomycin D Validates Modular Polyketide Synthase-Based Stereochemical Assignments.

Org Lett 2021 Oct 8;23(19):7439-7444. Epub 2021 Sep 8.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, U.K.

The stambomycins are a family of bioactive macrolides isolated from . Aside from two stereocenters installed through cytochrome P450 oxidations, their stereochemistry has been predicted by sequence analysis of the polyketide synthase. We report a synthesis of the C1-C27 fragment of stambomycin D, the spectroscopic data of which correlates well with that of the natural product, further validating predictive sequence analysis as a powerful tool for stereochemical assignment of complex polyketide natural products.
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http://dx.doi.org/10.1021/acs.orglett.1c02650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8491158PMC
October 2021

Impact of sulfamethoxazole on a riverine microbiome.

Water Res 2021 Aug 24;201:117382. Epub 2021 Jun 24.

University of Warwick, School of Life Sciences, Coventry, UK.

The continued emergence of bacterial pathogens presenting antimicrobial resistance is widely recognised as a global health threat and recent attention focused on potential environmental reservoirs of antibiotic resistance genes (ARGs). Freshwater environments such as rivers represent a potential hotspot for ARGs and antibiotic resistant bacteria as they are receiving systems for effluent discharges from wastewater treatment plants (WWTPs). Effluent also contains low levels of different antimicrobials including antibiotics and biocides. Sulfonamides are antibacterial chemicals widely used in clinical, veterinary and agricultural settings and are frequently detected in sewage sludge and manure in addition to riverine ecosystems. The impact of such exposure on ARG prevalence and diversity is unknown, so the aim of this study was to investigate the release of a sub-lethal concentration of the sulfonamide compound sulfamethoxazole (SMX) on the river bacterial microbiome using a flume system. This system was a semi-natural in vitro flume using river water (30 L) and sediment (6 kg) with circulation to mimic river flow. A combination of 'omics' approaches were conducted to study the impact of SMX exposure on the microbiomes within the flumes. Metagenomic analysis showed that the addition of low concentrations of SMX (<4 μg L) had a limited effect on the bacterial resistome in the water fraction only, with no impact observed in the sediment. Metaproteomics did not show differences in ARGs expression with SMX exposure in water. Overall, the river bacterial community was resilient to short term exposure to sub-lethal concentrations of SMX which mimics the exposure such communities experience downstream of WWTPs throughout the year.
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http://dx.doi.org/10.1016/j.watres.2021.117382DOI Listing
August 2021

Molecular basis for control of antibiotic production by a bacterial hormone.

Nature 2021 02 3;590(7846):463-467. Epub 2021 Feb 3.

Department of Chemistry, University of Warwick, Coventry, UK.

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
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http://dx.doi.org/10.1038/s41586-021-03195-xDOI Listing
February 2021

Phytoplankton trigger the production of cryptic metabolites in the marine actinobacterium Salinispora tropica.

Microb Biotechnol 2021 01 5;14(1):291-306. Epub 2020 Dec 5.

School of Life Sciences, University of Warwick, Coventry, UK.

Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co-culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria.
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http://dx.doi.org/10.1111/1751-7915.13722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888443PMC
January 2021

Corrigendum: Radiomic-Based Quantitative CT Analysis of Pure Ground-Glass Nodules to Predict the Invasiveness of Lung Adenocarcinoma.

Front Oncol 2020 30;10:608365. Epub 2020 Oct 30.

Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

[This corrects the article DOI: 10.3389/fonc.2020.00872.].
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http://dx.doi.org/10.3389/fonc.2020.608365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664516PMC
October 2020

Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management.

Sci Rep 2020 11 11;10(1):19566. Epub 2020 Nov 11.

School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.

Secoiridoid glycosides are anti-feeding deterrents of the Oleaceae family recently highlighted as potential biomarkers in Danish ash trees to differentiate between those tolerant and susceptible to the fungal disease ash dieback. With the knowledge that emerald ash borer has recently entered Europe from Russia, and that extensive selection trials are ongoing in Europe for ash dieback tolerant European ash (Fraxinus excelsior), we undertook comprehensive screening of secoiridoid glycosides in leaf extracts of trees tolerant and susceptible to ash dieback sampled from sites in the UK and Denmark. Here we report an unexpected diversity of secoiridoid glycosides in UK trees and higher levels of secoiridoid glycosides in the UK sample group. While it is unlikely that secoiridoid glycosides generally can serve as reliable markers for ash dieback susceptibility, there are differences between tolerant and susceptible groups for specific secoiridoids. We predict that the high levels-and structural diversity-of secoiridoids present in the UK group may provide a robust reservoir of anti-feeding deterrents to mitigate future herbivore threats such as the Emerald ash borer.
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http://dx.doi.org/10.1038/s41598-020-76140-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658974PMC
November 2020

Transferred by exosomes-derived MiR-19b-3p targets PTEN to regulate esophageal cancer cell apoptosis, migration and invasion.

Biosci Rep 2020 11;40(11)

Department of Thoracic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China.

The present study aims to investigate the relationship between miR-19b-3p and esophageal cancer (ESCA), and to detect the effects of miR-19b-3p transferred by exosomes on the phenotype of EC9706 cells. The expression of miR-19b-3p was detected by starBase analysis and real-time quantitative PCR (RT-qPCR). The target genes of miR-19b-3p were predicted by TargetScan and further verified by luciferase analysis. The mRNA and protein expression levels of PTEN and EMT-related genes were detected by RT-qPCR and Western blotting. The effects of miR-19b-3p transferred by exosomes and its target genes on the apoptosis, migration and invasion of EC9706 cells were studied by establishing a co-culture model of donor cells. The expression of miR-19b-3p in ESCA plasma, cells and exosomes was significantly up-regulated. miR-19b-3p transferred by exosomes could significantly reduce EC9706 cells apoptosis rate, promote cell migration and invasion, and could target the inhibition of PTEN expression. PTEN overexpression promoted apoptosis, inhibited cell migration and invasion, down-regulated the expression of MMP-2 and vimentin, and up-regulated E-cadherin expression; however, these effects could be partially reversed by miR-19b-3p. In summary, our results reveal that miR-19b-3p transferred by exosomes can target PTEN to regulate ESCA biological functions in the receptor EC9706 cells.
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http://dx.doi.org/10.1042/BSR20201858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685012PMC
November 2020

MmfL catalyses formation of a phosphorylated butenolide intermediate in methylenomycin furan biosynthesis.

Chem Commun (Camb) 2020 Nov;56(92):14443-14446

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.

Using a combination of a synthetic substrate analogue and product standard, MmfL, a homologue of the γ-butyrolactone biosynthetic enzyme AfsA, was shown to catalyse the condensation of dihydroxyacetone phosphate with a β-ketoacyl thioester to form a phosphorylated butenolide intermediate in the biosynthesis of the methylenomycin furans, which induce methlenomycin antibiotic production in Streptomyces coelicolor A3(2). AfsA homologues are also involved in the biosynthesis of 2-akyl-4-hydroxy-3-methyl butenolide inducers of antibiotic production in other Streptomyces species, indicating that diverse signalling molecules are assembled from analogous phosphorylated butenolide intermediates.
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http://dx.doi.org/10.1039/d0cc05658hDOI Listing
November 2020

Radiomic-Based Quantitative CT Analysis of Pure Ground-Glass Nodules to Predict the Invasiveness of Lung Adenocarcinoma.

Front Oncol 2020 11;10:872. Epub 2020 Aug 11.

Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

To investigate the performance of radiomic-based quantitative analysis on CT images in predicting invasiveness of lung adenocarcinoma manifesting as pure ground-glass nodules (pGGNs). A total of 275 lung adenocarcinoma cases, with 322 pGGNs resected surgically and confirmed pathologically, from January 2015 to October 2017 were enrolled in this retrospective study. All nodules were split into training and test cohorts randomly with a ratio of 4:1 to establish models to predict between pGGN-like adenocarcinoma (AIS)/minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IVA). Radiomic feature extraction was performed using Pyradiomics with semi-automatically segmented tumor regions on CT scans that were contoured with an in-house plugin for 3D-Slicer. Random forest (RF) and support vector machine (SVM) were used for feature selection and predictive model building in the training cohort. Three different predictive models containing conventional, radiomic, and combined models were built on the basis of the selected clinical, radiological, and radiomic features. The predictive performance of each model was evaluated through the receiver operating characteristic curve (ROC) and the area under the curve (AUC). The predictive performance of two radiologists (A and B) and our radiomic predictive model were further investigated in the test cohort to see if radiomic predictive model could improve radiologists' performance in prediction between pGGN-like AIS/MIA and IVA. Among 322 nodules, 48 (14.9%) were AIS and 102 (31.7%) were MIA with 172 (53.4%) for IVA. Age, diameter, density, and nine meaningful radiomic features were selected for model building in the training cohort. Three predictive models showed good performance in prediction between pGGN-like AIS/MIA and IVA (AUC > 0.8, < 0.05) in both training and test cohorts. The AUC values in the test cohort were 0.824 (95% CI, 0.723-0.924), 0.833 (95% CI, 0.733-0.934), and 0.848 (95% CI, 0.750-0.946) for conventional, radiomic, and combined models, respectively. The predictive accuracy was 73.44 and 59.38% for radiologist A and radiologist B in the test cohort and was improved dramatically to 79.69 and 75.00% with the aid of our radiomic predictive model. The predictive models built in our study showed good predictive power with good accuracy and sensitivity, which provided a non-invasive, convenient, economic, and repeatable way for the prediction between IVA and AIS/MIA representing as pGGNs. The radiomic predictive model outperformed two radiologists in predicting pGGN-like AIS/MIA and IVA, and could significantly improve the predictive performance of the two radiologists, especially radiologist B with less experience in medical imaging diagnosis. The selected radiomic features in our research did not provide more useful information to improve the combined predictive model's performance.
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http://dx.doi.org/10.3389/fonc.2020.00872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432133PMC
August 2020

Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin.

Microb Cell Fact 2020 May 24;19(1):111. Epub 2020 May 24.

Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), University of Oviedo, Oviedo, Spain.

Background: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear.

Results: Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps.

Conclusions: We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7-C12), followed by formation of the second and third rings (C5-C14; C3-C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1-C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.
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http://dx.doi.org/10.1186/s12934-020-01368-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247220PMC
May 2020

Bovistol B, bovistol D and strossmayerin: Sesquiterpene metabolites from the culture filtrate of the basidiomycete Coprinopsis strossmayeri.

PLoS One 2020 6;15(4):e0229925. Epub 2020 Apr 6.

School of Biological Sciences, University of Bristol, Bristol, United Kingdom.

Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B (1) and D (2), and one monomeric sesquiterpene, strossmayerin (7), were isolated from the culture filtrate of the basidiomycete fungus Coprinopsis strossmayeri. The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1-11 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135263PMC
July 2020

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal.

Nat Microbiol 2020 06 6;5(6):821-829. Epub 2020 Apr 6.

Department of Biology, Lund University, Lund, Sweden.

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB), which humans and various animals sense at extremely low concentrations. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%), suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.
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http://dx.doi.org/10.1038/s41564-020-0697-xDOI Listing
June 2020

An investigation and validation of CT scan in detection of spinal epidural adipose tissue.

Medicine (Baltimore) 2020 Mar;99(10):e19448

School of Statistics and Mathematics, Zhejiang Gongshang University, Hangzhou, PR China.

To investigate the accuracy of computed tomography (CT) in evaluating spinal epidural adipose tissue compared to magnetic resonance imaging (MRI).CT scan images and matched magnetic resonance images of total 368 patients between July 2014 and July 2016 were evaluated. Hounsfield units (HU) of epidural fat (EF), dural sac (DuS), ligamentum flavum, bone of facet joints, and paraspinal muscles were measured for comparison. Anteroposterior diameter of the EF, anteroposterior diameter of the DuS, transverse diameter of the DuS, cross-sectional area of the EF, and cross-sectional area of the DuS were measured at each disc level from L1-2 to L5-S1.Fat tissue showed exclusive negative HU significantly different from all other periphery tissues. Pearson correlation coefficient analyses showed significant positive correlations between CT and MRI measurements; Bland-Altman plots also depicted satisfied agreement. Overgrowth of spinal EF was more commonly found at L2-3 and L3-4 levels in present study, and body weight, age, and gender were significantly associated with amounts of EF both on CT and MRI.The CT scan is a satisfied alternative of MRI for the evaluation of spinal epidural adipose tissue.
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http://dx.doi.org/10.1097/MD.0000000000019448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478604PMC
March 2020

Pentamycin Biosynthesis in Philippine Streptomyces sp. S816: Cytochrome P450-Catalyzed Installation of the C-14 Hydroxyl Group.

ACS Chem Biol 2019 06 31;14(6):1305-1309. Epub 2019 May 31.

Department of Chemistry , University of Warwick , Coventry CV4 7AL , United Kingdom.

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis, and mixed infections. Chemical instability has hindered its widespread application and development as a drug. Here, we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species.
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http://dx.doi.org/10.1021/acschembio.9b00270DOI Listing
June 2019

A trans-Acting Cyclase Offloading Strategy for Nonribosomal Peptide Synthetases.

ACS Chem Biol 2019 05 8;14(5):845-849. Epub 2019 Apr 8.

Department of Chemistry , University of Warwick , Coventry CV4 7AL , United Kingdom.

The terminal step in the biosynthesis of nonribosomal peptides is the hydrolytic release and, frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The surugamide biosynthetic pathway is composed of two nonribosomal peptide synthetase (NRPS) assembly lines in which one produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to question how the peptides are released from the assembly line (and cyclized in the case of surugamide A). We characterized a cyclase belonging to the β-lactamase superfamily in vivo, established that it is a trans-acting release factor for both compounds, and verified this functionality in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ∼11% of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead employ a trans-acting cyclase. This study improves the paradigmatic understanding of how nonribosomal peptides are released from the terminal peptidyl carrier protein and adds a new dimension to the synthetic biology toolkit.
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http://dx.doi.org/10.1021/acschembio.9b00095DOI Listing
May 2019

Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery.

Chem Sci 2019 Jan 19;10(2):453-463. Epub 2018 Oct 19.

Warwick Integrative Synthetic Biology Centre and School of Life Sciences , University of Warwick , Coventry , CV4 7AL , UK . Email: ; Email:

In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against , as well as inhibition of the cancer-associated metabolic enzyme nicotinamide -methyltransferase (NNMT).
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http://dx.doi.org/10.1039/c8sc03814gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335953PMC
January 2019

Flavopiridol Protects Bone Tissue by Attenuating RANKL Induced Osteoclast Formation.

Front Pharmacol 2018 3;9:174. Epub 2018 May 3.

Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

Bone resorption and homeostasis is carried out by osteoclasts, whose differentiation and activity are regulated by the RANK/RANKL axis. Our previous studies using a mouse model of joint injury show that joint trauma induces local inflammation followed by bone remodeling. The transcription factor cyclin-dependent kinase 9 (CDK9) is the major regulator of inflammation, as CDK9 inhibitor flavopiridol effectively suppress injury-induced inflammatory response. The objective of this study was to investigate the underlying mechanism through which flavopiridol regulates bone resorption. The effects of CDK9 inhibition, by the specific-inhibitor flavopiridol, on bone resorption were determined using two distinct and clinically relevant bone remodeling models. The first model involved titanium particle-induced acute osteolysis, and the second model was ovariectomy-induced chronic osteoporosis. The effects and mechanism of CDK9 inhibition on osteoclastogenesis were examined using culture of bone marrow macrophages (BMMs). Our results indicated that flavopiridol potently suppressed bone resorption in both bone-remodeling models. In addition, CDK9 inhibition suppressed osteoclastogenesis of BMM and reduced their expression of osteoclast-specific genes. Finally, we determined that flavopiridol suppressed RANKL signaling pathway via inhibition of p65 phosphorylation and nuclear translocation of NF-κB. Summary, CDK9 is a potential therapeutic target to prevent osteolysis and osteoporosis by flavopiridol treatment.
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http://dx.doi.org/10.3389/fphar.2018.00174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944179PMC
May 2018

New activation mechanism for half-sandwich organometallic anticancer complexes.

Chem Sci 2018 Mar 1;9(12):3177-3185. Epub 2018 Mar 1.

Department of Chemistry , University of Warwick , Gibbet Hill Road , Coventry CV4 7AL , UK . Email:

The Cp C-H protons in certain organometallic Rh half-sandwich anticancer complexes [(η-Cp )Rh(,')Cl], where Cp = Cp*, phenyl or biphenyl-MeCp, and ,' = bipyridine, dimethylbipyridine, or phenanthroline, can undergo rapid sequential deuteration of all 15 Cp* methyl protons in aqueous media at ambient temperature. DFT calculations suggest a mechanism involving abstraction of a Cp* proton by the Rh-hydroxido complex, followed by sequential H/D exchange, with the Cp* rings behaving like dynamic molecular 'twisters'. The calculations reveal the crucial role of p orbitals of ,'-chelated ligands in stabilizing deprotonated Cp ligands, and also the accessibility of Rh-fulvene intermediates. They also provide insight into why biologically-inactive complexes such as [(Cp*)Rh(en)Cl] and [(Cp*)Ir(bpy)Cl] do not have activated Cp* rings. The thiol tripeptide glutathione (γ-l-Glu-l-Cys-Gly, GSH) and the activated dienophile -methylmaleimide, (NMM) did not undergo addition reactions with the proposed Rh-fulvene, although they were able to control the extent of Cp* deuteration. We readily trapped and characterized Rh-fulvene intermediates by Diels-Alder [4+2] cyclo-addition reactions with the natural biological dienes isoprene and conjugated (9,11)-linoleic acid in aqueous media, including cell culture medium, the first report of a Diels-Alder reaction of a metal-bound fulvene in aqueous solution. These findings will introduce new concepts into the design of organometallic Cp* anticancer complexes with novel mechanisms of action.
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http://dx.doi.org/10.1039/c7sc05058eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916112PMC
March 2018

NMR-based assignment of isoleucine vs. allo-isoleucine stereochemistry.

Org Biomol Chem 2017 Nov;15(44):9372-9378

Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.

A simple H and C NMR spectrometric analysis is demonstrated that permits differentiation of isoleucine and allo-isoleucine residues by inspection of the chemical shift and coupling constants of the signals associated with the proton and carbon at the α-stereocentre. This is applied to the estimation of epimerisation during metal-free N-arylation and peptide coupling reactions.
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http://dx.doi.org/10.1039/c7ob01995eDOI Listing
November 2017

Quantification of the antimalarial drug pyronaridine in whole blood using LC-MS/MS - Increased sensitivity resulting from reduced non-specific binding.

J Pharm Biomed Anal 2017 Nov 26;146:214-219. Epub 2017 Aug 26.

Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand; Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK.

Malaria is one of the most important parasitic diseases of man. The development of drug resistance in malaria parasites is an inevitable consequence of their widespread and often unregulated use. There is an urgent need for new and effective drugs. Pyronaridine is a known antimalarial drug that has received renewed interest as a partner drug in artemisinin-based combination therapy. To study its pharmacokinetic properties, particularly in field settings, it is necessary to develop and validate a robust, highly sensitive and accurate bioanalytical method for drug measurements in biological samples. We have developed a sensitive quantification method that covers a wide range of clinically relevant concentrations (1.5ng/mL to 882ng/mL) using a relatively low volume sample of 100μL of whole blood. Total run time is 5min and precision is within ±15% at all concentration levels. Pyronaridine was extracted on a weak cation exchange solid-phase column (SPE) and separated on a HALO RP amide fused-core column using a gradient mobile phase of acetonitrile-ammonium formate and acetonitrile-methanol. Detection was performed using electrospray ionization and tandem mass spectrometry (positive ion mode with selected reaction monitoring). The developed method is suitable for implementation in high-throughput routine drug analysis, and was used to quantify pyronaridine accurately for up to 42days after a single oral dose in a drug-drug interaction study in healthy volunteers.
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http://dx.doi.org/10.1016/j.jpba.2017.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637160PMC
November 2017

Watasemycin biosynthesis in : thiazoline C-methylation by a type B radical-SAM methylase homologue.

Chem Sci 2017 Apr 19;8(4):2823-2831. Epub 2017 Jan 19.

Department of Chemistry , University of Warwick , Coventry , CV4 7AL , UK . Email:

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive species. Transcriptional analyses of ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in , was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis.
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http://dx.doi.org/10.1039/c6sc03533gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427693PMC
April 2017

Role of GntR Family Regulatory Gene in Gluconate Metabolism in M145.

Biomed Res Int 2017 27;2017:9529501. Epub 2017 Apr 27.

Department of Genetics and Biotechnology, Ivan Franko National University of Lviv, Lviv 79005, Ukraine.

Here we report functional characterization of the M145 gene which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that is part of putative operon involved in gluconate metabolism. Combining the results of knockout, transcriptional analysis of operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes and . The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by . In comparison to the wild type, on gluconate-containing minimal medium, the mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.
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http://dx.doi.org/10.1155/2017/9529501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425828PMC
March 2018

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

J Am Chem Soc 2017 06 5;139(23):7974-7981. Epub 2017 Jun 5.

Organisms and Environment Research Division, Cardiff School of Biosciences, Cardiff University , Cardiff CF10 3AT, United Kingdom.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.
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http://dx.doi.org/10.1021/jacs.7b03382DOI Listing
June 2017

A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units.

Nat Commun 2016 12 21;7:13609. Epub 2016 Dec 21.

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.
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http://dx.doi.org/10.1038/ncomms13609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187497PMC
December 2016

Phytoremediation-biorefinery tandem for effective clean-up of metal contaminated soil and biomass valorisation.

Int J Phytoremediation 2017 Nov;19(11):965-975

a Warwick Manufacturing Group , University of Warwick , Coventry , UK.

During the last few decades, phytoremediation process has attracted much attention because of the growing concerns about the deteriorating quality of soil caused by anthropogenic activities. Here, a tandem phytoremediation/biorefinery process was proposed as a way to turn phytoremediation into a viable commercial method by producing valuable chemicals in addition to cleaned soil. Two agricultural plants (Sinapis alba and Helianthus annuus) were grown in moderately contaminated soil with ca. 100 ppm of Ni and further degraded by a fungal lignin degrader-Phanerochaete chrysosporium. Several parameters have been studied, including the viability of plants, biomass yield, and their accumulating and remediating potentials. Further, downstream processing showed that up to 80% of Ni can be easily extracted from contaminated biomass by aqueous extraction at mild conditions. Finally, it was demonstrated that the growth of plants on the contaminated soil could be degraded by P. chrysosporium, and the effect of nickel and biomass pretreatment on the solid-state fermentation was studied. The proposed and studied methodology in this work could pave the way for successful commercialization of the phytoremediation process in the near future.
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http://dx.doi.org/10.1080/15226514.2016.1267705DOI Listing
November 2017

Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster.

Chembiochem 2016 Nov 13;17(22):2189-2198. Epub 2016 Oct 13.

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one-vemR-that encodes a transcriptional activator of the large ATP-binding LuxR-like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co-expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin-producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.
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http://dx.doi.org/10.1002/cbic.201600396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132015PMC
November 2016

Second-generation probes for biosynthetic intermediate capture: towards a comprehensive profiling of polyketide assembly.

Chem Commun (Camb) 2016 Aug;52(68):10392-5

Department of Chemistry, University of Warwick, Library Road, CV4 7AL, UK.

Malonyl carba(dethia) N-decanoyl cysteamine methyl esters and novel acetoxymethyl esters were utilised as second-generation probes for polyketide intermediate capture. The use of these tools in vivo led to the characterisation of an almost complete set of biosynthetic intermediates from a modular assembly line, providing a first kinetic overview of intermediate processing leading to complex natural product formation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050551PMC
http://dx.doi.org/10.1039/c6cc04681aDOI Listing
August 2016

Insights into 6-Methylsalicylic Acid Bio-assembly by Using Chemical Probes.

Angew Chem Weinheim Bergstr Ger 2016 03 2;128(10):3524-3528. Epub 2016 Feb 2.

Department of Chemistry University of Warwick Library Road Coventry CV4 7AL UK.

Chemical probes capable of reacting with KS (ketosynthase)-bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6-methylsalicylic acid synthase (6-MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6-MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6-MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.
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http://dx.doi.org/10.1002/ange.201509038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4950124PMC
March 2016

Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152.

Org Biomol Chem 2016 Jul;14(27):6390-3

Department of Chemistry and School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.

Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8. Incorporation experiments using isotopically-labelled precursors confirmed the chemical structures of SCB1-3 and established those of SCB4-8.
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http://dx.doi.org/10.1039/c6ob00840bDOI Listing
July 2016
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