Crombie, Andrew (2011) Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2. PhD thesis, University of Warwick.

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Abstract

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph
isolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range
of multi-carbon compounds in addition to methane. An analysis of growth conditions
identified the requirements for robust and predictable growth on a wide range of
substrates. A simple and effective method of targeted mutagenesis was developed,
which relies on electroporation with a linear DNA fragment, and several strains with
deletions of key enzymes were constructed using this method. Deletion of isocitrate
lyase demonstrated that this enzyme is required for growth on both one-carbon and
two-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase,
was shown to be essential for growth on two-carbon compounds. However,
surprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on
expression of methanol dehydrogenase. Possible causes of this effect are discussed.
Surprisingly, M. silvestris was able to grow on propane and the presence and
expression of a gene cluster encoding a putative propane monooxygenase was
confirmed. This enzyme was found to be a second soluble diiron monooxygenase
(SDIMO) with homology to the propane monooxygenase from Gordonia TY5,
identifying M. silvestris as the first known methanotroph to contain SDIMOs from
more than one group. Deletion of these enzymes in turn was used to determine the
requirement for each during growth on methane or propane. The soluble methane
monooxygenase (sMMO) was found to be capable of oxidising propane, whereas the
propane monooxygenase (PrMO) was unable to oxidise methane. However, although
a strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was
unable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was
less efficient in comparison to the wild-type. Evidence is presented that products of
oxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in
the absence of the PrMO. Both the sMMO and the PrMO were found to be capable of
oxidation of a wide range of aliphatic and aromatic compounds, including
xenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was
found to oxidise propane at both terminal and sub-terminal positions, resulting in 1-
propanol and 2-propanol respectively, and biochemical methods were used to assay
the enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was
found to be by the methylmalonyl-CoA pathway, and the data suggested that 2-
propanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster,
predicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme
with homology to characterised membrane-bound D-gluconate dehydrogenase from
Gluconobacter spp., was found to be essential for growth on 2-propanol and acetone
and may be involved in the oxidation of acetol during propane metabolism by the
sub-terminal pathway.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Methanotrophs -- Growth, Methanotrophs -- Metabolism, Methanotrophs -- Genetics, Mutagenesis, Propane
Official Date:	September 2011
Dates:	Date Event September 2011 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Murrell, Colin
Extent:	xxiii, 291 leaves : ill., charts
Language:	eng

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