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Towards an effective biosensor for monitoring AD leachate: a knockout E. coli mutant that cannot catabolise lactate

2015-12, Sweeney, Joseph, Murphy, Cormac D., McDonnell, Kevin

Development of a biosensor for the convenient measurement of acetate and propionate concentrations in a two-phase anaerobic digestor (AD) requires a bacterium that will be unresponsive to the other organic acids present in the leachate, of which lactate is the most abundant. Successive gene knockouts of E.coli W3110 d-lactate dehydrogenase (dld), l-lactate dehydrogenase (lldD), glycolate oxidase (glcD) and a suspected l-lactate dehdrogenase (ykgF) were performed. The resulting quadruple mutant (IMD Wldgy) was incapable of growth on d- and l-lactate, whereas the wild type grew readily on these substrates. Furthermore, the O2 consumption rates of acetate-grown IMD Wldgy cell suspensions supplied with either acetate (0.1 mM) or a synthetic leachate including acetate (0.1 mM) and dl-lactate (1 mM) were identical (2.79 and 2.70 mg l−1 min−1, respectively). This was in marked contrast to similar experiments with the wild type which gave initial O2 consumption rates of 2.00, 2.36 and 2.97 mg l−1 min−1 when cell suspensions were supplied with acetate (0.1 mM), acetate (0.1 mM) plus d-lactate (1 mM) or acetate (0.1 mM) plus l-lactate (1 mM), respectively. The knockout strain provides a platform for the design of a biosensor that can accessibly monitor acetate and propionate concentrations in AD leachate via O2-uptake measurements.

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Biosynthesis of pyrrolylpolyenes in Auxarthron umbrinum

2008-10-31, Clark, Benjamin R., Murphy, Cormac D.

The biosynthesis of the pyrrolylpolyene rumbrin (1) in the fungus Auxarthron umbrinum was elucidated using feeding studies with labelled precursors. Incorporation of stable isotopes from [15N]-proline, [13C]-methionine and [13C]-acetate confirmed that these were the precursors of the pyrrole moiety, methyl groups, and backbone of rumbrin, respectively. Label-dilution experiments with pyrrole-2-carboxylate confirmed it was a direct precursor in the biosynthesis of rumbrin. Both 3- and 4-chloropyrrolecarboxylates were also accepted as precursors in polyene production.

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A convenient chemical-microbial method for developing fluorinated pharmaceuticals

2013, Bright, Tara V., Dalton, Fay, Elder, Victoria L., Murphy, Cormac D., O'Connor, Neil K., Sandford, Graham

A significant proportion of pharmaceuticals are fluorinated and selecting the site of fluorine incorporation can be an important beneficial part a drug development process. Here we describe initial experiments aimed at the development of a general method of selecting optimum sites on pro - drug molecules for fluorination, so that metabolic stability may be improved. Several model biphenyl derivatives were transformed by the fungus Cunninghamella elegans and the bacterium Streptomyces griseus, both of which contain cytochromes P450 that mimic oxidation processes in vivo, so that the site of oxidation could be determined. Subsequently, fluorinated biphenyl derivatives were synthesised using appropriate Suzuki - Miyaura coupling reactions, positioning the fluorine atom at the pre - determined site of microbial oxidation; the fluorinated biphenyl derivatives were incubated with the microorganisms and the degree of oxidation assessed. Biphenyl-4-carboxylic acid was transformed completely to 4' - hydroxybiphenyl - 4 - carboxylic acid by C. elegans but, in contrast, the 4' fluoro - analogue remained untransformed exemplifying the microbial oxidation – chemical fluorination concept. 2' - Fluoro-and 3' - fluoro - biphenyl - 4 - carboxylic acid were also transformed, but more slowly than the non - fluorinated biphenyl carboxylic acid derivative. Thus, it is possible to design compounds in an iterative fashion with a longer metabolic half - life by identifying the sites that are most easily oxidised by in vitro methods and subsequent fluorination without recourse to extensive animal studies.

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Biotransformation of fluorobiphenyl by Cunninghamella elegans

2010, Amadio, Jessica, Murphy, Cormac D.

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by 19F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.

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Precursor-directed biosynthesis of fluorinated iturin A in Bacillus spp.

2009, Moran, Stephen, Rai, Dilip K., Clark, Benjamin R., Murphy, Cormac D.

Some iturin A-producing strains of Bacillus subtilis will elaborate the novel fluorinated analogue when incubated with 3-fluoro-L-tyrosine. The activity of iturin A is dependent on the D-tyrosine residue and the presence of fluorotyrosine may result in an improvement of the biological properties of this lipopeptide. The fluorinated iturin might also be used as a probe for studying its interaction with biological membranes.

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Biodegradation of polyfluorinated biphenyl in bacteria

2011-07, Hughes, David, Clark, Benjamin R., Murphy, Cormac D.

Fluorinated aromatic compounds are significant environmental pollutants, and microorganisms play important roles in their biodegradation. The effect of fluorine substitution on the transformation of fluorobiphenyl in two bacteria was investigated. Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400 used 2,3,4,5,6-pentafluorobiphenyl and 4,4′-difluorobiphenyl as sole sources of carbon and energy. The catabolism of the fluorinated compounds was examined by gas chromatography–mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), and revealed that the bacteria employed the upper pathway of biphenyl catabolism to degrade these xenobiotics. The novel fluorometabolites 3-pentafluorophenyl-cyclohexa-3,5-diene-1,2-diol and 3-pentafluorophenyl-benzene-1,2-diol were detected in the supernatants of biphenyl-grown resting cells incubated with 2,3,4,5,6-pentafluorobiphenyl, most likely as a consequence of the actions of BphA and BphB. 4-Fluorobenzoate was detected in cultures incubated with 4,4′-difluorobiphenyl and 19F NMR analysis of the supernatant from P. pseudoalcaligenes KF707 revealed the presence of additional water-soluble fluorometabolites.

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Exploiting the genome sequence of Streptomyces nodosus for enhanced antibiotic production

2016-02, Sweeney, Paul, Murphy, Cormac D., Caffrey, Patrick

The genome of the amphotericin producer Streptomyces nodosus was sequenced. A single scaffold of 7,714,110 bp was obtained. Biosynthetic genes were identified for several natural products including polyketides, peptides, siderophores and terpenes. The majority of these clusters specified known compounds. Most were silent or expressed at low levels and unlikely to compete with amphotericin production. Biosynthesis of a skyllamycin analogue was activated by introducing expression plasmids containing either a gene for a LuxR transcriptional regulator or genes for synthesis of the acyl moiety of the lipopeptide. In an attempt to boost amphotericin production, genes for acyl CoA carboxylases, a phosphopantetheinyl transferase and the AmphRIV transcriptional activator were overexpressed, and the effects on yields were investigated. This study provides the groundwork for metabolic engineering of S. nodosus strains to produce high yields of amphotericin analogues.

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Drug metabolism in microorganisms

2015-01, Murphy, Cormac D.

Several wild type and recombinant microorganisms can transform drugs to the equivalent human metabolites. Fungi, such as Cunninghamella spp., and Streptomyces bacteria express cytochrome P450 (CYP) enzymes that enable analogous phase I (oxidative) reactions with a wide range of drugs. The gene encoding the bifunctional CYP102A1 in Bacillus megaterium can be expressed easily in E. coli, and extensive mutagenesis experiments have generated numerous variants that can produce human drug metabolites. Additionally, human CYP isoforms have been expressed in various hosts. The application of microbial CYPs to the production of human drug metabolites is reviewed, and additional applications in the field of drug development are considered.

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New insights into polyene macrolide biosynthesis in Couchioplanes caeruleus

2017-05-01, Sheehan, James, Murphy, Cormac D., Caffrey, Patrick

Couchioplanes caeruleus DSM43634 synthesises 67–121C, an aromatic heptaene macrolide that contains a mannosyl-mycosaminyl disaccharide. An improved draft genome sequence was used to obtain the biosynthetic gene cluster for this antifungal. Bioinformatic analysis of the polyketide synthase indicated that extension modules 7 and 8 contain A-type ketoreductase and dehydratase domains. These modules are therefore predicted to form cis double bonds. The deduced stereostructure of the 67–121C macrolactone is identical to that experimentally determined for the partricin subgroup of aromatic heptaenes. Some of these polyenes are N-methylated on the aminoacetophenone moiety. The C. caeruleus AceS protein was shown to methylate 4-aminoacetophenone and esters of 4-aminobenzoate, but not 4-aminobenzoate. This suggests that the substrate specificity of AceS prevents it from interfering with folate biosynthesis. The methyltransferase should be valuable for chemoenzymatic alkylation of compounds that contain aminobenzoyl moieties.

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Treatment of fluoroacetate by a Pseudomonas fluorescens biofilm grown in membrane aerated biofilm reactor

2009, Heffernan, Barry, Murphy, Cormac D., Syron, Eoin, Casey, Eoin

Fluorinated organic compounds have widespread applications, and their accumulation in the environment is a concern. Biofilm reactors are an effective technology for the treatment of contaminated wastewater, yet almost no research has been conducted on the effectiveness of biofilms for the biodegradation of fluorinated aliphatic compounds. In this paper we describe experiments undertaken to investigate the degradation of fluoroacetate using a membrane aerated biofilm reactor (MABR) by Pseudomonas fluorescens DSM8341. The concentration of fluoroacetate in the medium influenced biofilm structure, with less dense biofilm observed at lower fluoroacetate loading rates. As biofilm thickness increased, oxygen utilization decreased, probably as a consequence of increased resistance to oxygen transfer. Furthermore, most of the biofilm was anaerobic, since oxygen penetration depth was less than 1000 μm. Biofilm performance, in terms of fluoroacetate removal efficiency, was improved by decreasing the fluoroacetate loading rate, however increasing the intramembrane oxygen pressure had little effect on biofilm performance. A mathematical model showed that while fluoroacetate does not penetrate the entire biofilm, the defluorination intermediate metabolite glycolate does, and consequently the biofilm was not carbon limited at the biofilm−membrane interface where oxygen concentrations were highest. The model also showed the accumulation of the free fluoride ion within the biofilm. Overflow metabolism of glycolate was identified to be most likely a result of a combination of oxygen limitation and free fluoride ion inhibition. The study demonstrated the potential of MABR for treating wastewater streams contaminated with organofluorine compounds.