Molecular and biochemical characterisation of isoprene metabolism by Variovorax sp. WS11

Dawson, Robin (2021) Molecular and biochemical characterisation of isoprene metabolism by Variovorax sp. WS11. Doctoral thesis, University of East Anglia.

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Abstract

Isoprene is the most abundant non-methane volatile organic compound (VOC), with over 500 Tg C yr-1 produced worldwide. Terrestrial plants, particularly trees, are the main sources of isoprene, and soils are currently the largest known sink for atmospheric isoprene. The work described in this thesis aimed to characterise the isoprene metabolic pathway used by the novel Gram negative bacterium, Variovorax sp. WS11. Isoprene monooxygenase (IsoMO), the plasmid-encoded soluble diiron monooxygenase (SDIMO) first identified in Rhodococcus sp. AD45, was characterised in Variovorax sp. WS11 by targeted mutagenesis and heterologous expression of the IsoMO-encoding genes, isoABCDEF. Linear alkynes selectively inhibited isoprene oxidation by different SDIMO enzymes, with octyne inhibiting isoprene oxidation by IsoMO and acetylene inhibiting the co-oxidation of isoprene by the soluble methane monooxygenase (sMMO). These findings provided avenues for further studies of isoprene degrading communities in soils.

Analysis of the proteome of isoprene-grown Variovorax sp. WS11 allowed the prediction of the full isoprene metabolic pathway. Propionyl-CoA was predicted as a metabolic intermediate due to the significant expression of peptides which catalyse the methylcitrate cycle and the methylmalonyl-CoA pathway. These data indicated that Variovorax sp. WS11 incorporates isoprene-derived carbon via a different metabolic pathway than Rhodococcus sp. AD45, in which the genes encoding these pathways were not expressed during growth on isoprene. Further molecular and biochemical analyses are required to confirm the roles of the putative methylmalonyl-CoA and methylcitrate pathways in isoprene metabolism.

The expression of the iso metabolic gene cluster was regulated by two LysR-type transcriptional regulators, with targeted mutations indicating that dmlR_4 encoded a transcriptional activator and dmlR_5 encoded a transcriptional repressor. Isoprene metabolism was differentially regulated in the presence of alternative carbon sources, with glucose repressing the expression of the iso gene cluster and pyruvate stimulating expression of isoA and the apparent activity of IsoMO.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Chris White
Date Deposited: 23 Mar 2022 10:25
Last Modified: 23 Mar 2022 10:25
URI: https://ueaeprints.uea.ac.uk/id/eprint/84235
DOI:

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