Elucidation of molecular mechanisms of antibiotics biosynthesis in Burkholderia gladioli

Burkholderia is a multi-talented genus of Gram-negative bacteria that has been recently shown to be a promising, untapped source of antibiotics with the potential to overcome antimicrobial resistance. An antimicrobial activity screen of a clinical isolate, B. gladioli BCC0238, identified a novel polyketide macrolide antibiotic, gladiolin, that is structurally related to a known antibiotic etnangien and exhibits potent activity against Mycobacterium tuberculosis. Gladiolin shows significantly increased stability towards light and air compared to etnangien, due to the absence of a highly unstable hexaene moiety present in the side chain of etnangien, which is the key structural difference between the two metabolites. Comparison of the polyketide synthases (PKSs) responsible for the biosynthesis of gladiolin and etnangien, however, reveals a strikingly similar domain architecture. This thesis reports the elucidation of the catalytic origins for the main structural differences between the two metabolites, revealing a trans-acting enoyl reduction event involved in shutting down a programmed iteration in gladiolin biosynthesis, which is proposed for the polyene formation in etnangien biosynthesis. In addition to gladiolin, a set of lipopeptodiolides, known as icosalides, which were originally reported as fungal metabolites, were also discovered from B. gladioli BCC0238. The non-ribosomal peptide synthase (NRPS) responsible for the biosynthesis of icosalides exhibits an unprecedented domain architecture revealing two directly adjacent condensation (C) domains embedded in the middle of the NRPS. In this study, the enzymology of these C domains has been investigated in vitro, elucidating an unusual double chain initiation mechanism for asymmetric peptidolide biosynthesis. Additionally, efforts towards in vitro reconstitution of the entire icosalide NRPS is reported, which may allow access to novel antibiotic analogues

Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex.

Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.This work was supported by a project grant from the Medical Research Council, UK (G1001687) to P.F.L.; and by the 973 and 863 programs from the Ministry of Science and Technology of China, National Science Foundation of China, and the Translational Medical Research Fund of Wuhan University School of Medicine to Y.S.; E.M. thanks the Gates Cambridge Trust for a scholarship. We also gratefully acknowledge Dr. Xinzhou Yang, SouthCentral University for Nationalities, for his assistance in separation of gentamicin A2. We thank Dr. Andrew Truman (John Innes Institute) for helpful discussions.This is the final published version. It was originally published in Chemistry and Biology, Volume 22, Issue 2, 19 February 2015, Pages 251–261, doi:10.1016/j.chembiol.2014.12.01

Antibiotic skeletal diversification via differential enoylreductase recruitment and module iteration in trans -acyltransferase polyketide synthases

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the “blind watchmaker” of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons

Discovery and biosynthesis of bolagladins : unusual lipodepsipeptides from Burkholderia gladioli clinical isolates

wo Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate‐derived fatty acid and a rare dehydro‐β‐alanine residue. The gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In‐frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of about 50% of B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S (CAS) assay indicated that these metabolites bind ferric iron, which suppresses their production when added to the growth medium. Moreover, a gene encoding a TonB‐dependent ferric‐siderophore receptor is adjacent to the biosynthetic genes, suggesting that these metabolites may function as siderophores in B. gladioli

The histone deacetylase inhibitor tubacin mitigates endothelial dysfunction by up-regulating the expression of endothelial nitric oxide synthase.

Endothelial nitric oxide (NO) synthase (eNOS) plays a critical role in the maintenance of blood vessel homeostasis. Recent findings suggest that cytoskeletal dynamics play an essential role in regulating eNOS expression and activation. Here, we sought to test whether modulation of cytoskeletal dynamics through pharmacological regulation of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation affects eNOS expression and endothelial function in vitro and in vivo.Wefound that tubulin acetylation inducer (tubacin), a compound that appears to selectively inhibit HDAC6 activity, dramatically increased eNOS expression in several different endothelial cell lines, as determined by both immunoblotting and NO production assays. Mechanistically, we found that these effects were not mediated by tubacin\u27s inhibitory effect on HDAC6 activity, but rather were due to its ability to stabilize eNOS mRNA transcripts. Consistent with these findings, tubacin also inhibited proinflammatory cytokine-induced degradation of eNOS transcripts and impairment of endothelium-dependent relaxation in the mouse aorta. Furthermore, we found that tubacin-induced up-regulation in eNOS expression in vivo is associated with improved endothelial function in diabetic db/db mice and with a marked attenuation of ischemic brain injury in a murine stroke model. Our findings indicate that tubacin exhibits potent eNOS-inducing effects and suggest that this compound might be useful for the prevention or management of endothelial dysfunction-associated cardiovascular diseases. © 2019 Chen et al

Towards interpreting recurrent neural networks through probabilistic abstraction

National Research Foundation (NRF) Singapore under its AI Singapore Programm

Antimicrobial Agents. PCT Int. Appl., 2018, WO/2018/193273

Patent Applicationstatus: publishe

The Impact of Yaw Attitude of Eclipsing GPS/GALILEO Satellites on Kinematic PPP Solutions and Their Correction Models

When GPS/GALILEO satellite runs to the position where it is approximately collinear with the sun and the earth, it is difficult for the satellite to keep nominal attitude, so it will show abnormal yaw attitude for a period of time. Based on the precision orbit and clock correction products offered by different analysis centers, we design different attitude correction strategies for satellite that is in abnormal yaw attitude period, select 10-day measured data from 7 MGEX stations distributed globally, and analyze the influence of antenna phase center offset and phase wind-up of GPS/GALILEO satellite on residuals of observations and kinematic PPP positioning result in this paper. The research results show that when the satellite is in abnormal yaw attitude period, adopting nominal yaw attitude can have an impact up to 8 cm and 11 cm on the residuals of GPS/GALILEO satellite observations. GPS/GALILEO satellite is in model yaw attitude during the period and its positioning accuracy of kinematic PPP positioning results in three directions of E, N and U shows an increasing rate of 13.30%, 15.77% and 12.98%, respectively in comparison with that in nominal yaw attitude. Comparing with satellite deletion strategy, the accuracy of kinematic PPP positioning results in three directions of E, N and U when the satellite is in model yaw attitude shows an increasing rate of 5.399%, 4.430% and 5.992%, respectively

Diene incorporation by a dehydratase domain variant in modular polyketide synthases

Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon–carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold

Video_2_Case report: Short-term efficacy and changes in 18F-FDG-PET with acute multi-target stimulation in spinocerebellar ataxia type 3 (SCA3/MJD).MP4

ObjectiveSpinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a rare neurodegenerative disease for which there is no specific treatment. Very few cases have been treated with single-target deep brain stimulation (DBS), and the results were not satisfactory. We applied multi-target DBS to an SCA3/MJD patient and performed positron emission computed tomography (PET) before and after DBS to explore the short-term clinical therapeutic effect.Materials and methodsA 26-year-old right-hand-dominant female with a family history of SCA3/MJD suffered from cerebellar ataxia and dystonia. Genetic testing indicated an expanded CAG trinucleotide repeat in the ATXN3 gene and a diagnosis of SCA3/MJD. Conservative treatment had no obvious effect; therefore, leads were implanted in the bilateral dentate nucleus (DN) and the globus pallidus internus (GPi) and connected to an external stimulation device. The treatment effect was evaluated in a double-blind, randomized protocol in five phases (over a total of 15 days): no stimulation, GPi, DN, or sham stimulation, and combined GPi and DN stimulation. 18F-fluoro-2-deoxy-d-glucose and dopamine transporter PET, Scale for the Assessment and Rating of Ataxia, Fahn-Tolosa-Marin Clinical Rating Scale for Tremor (FTM), Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS), and SF-36 quality of life scores were compared before and after DBS.ResultsThe Total Scale for the Assessment and Rating of Ataxia scores improved by ~42% (from 24 to 14). The BFMDRS movement scores improved by ~30% (from 40.5 to 28.5). The BFMDRS disability scores improved by ~12.5% (from 16 to 14). Daily living activities were not noticeably improved. Compared with the findings in pre-DBS imaging, 18F-fluoro-2-deoxy-d-glucose uptake increased in the cerebellum, while according to dopamine transporter imaging, there were no significant differences in the bilateral caudate nucleus and putamen.ConclusionMulti-target acute stimulation (DN DBS and GPi DBS) in SCA3/MJD can mildly improve cerebellar ataxia and dystonia and increase cerebellar metabolism.</p