Smith, Thomas J. and Murrell, J. C. (J. Colin) (2011) Mutagenesis of soluble methane monoozygenase. In: Methods in methane metabolism. Part B , Methanotrophy. Methods in enzymology, Vol.495 . Elsevier/Academic Press, Amsterdam, [Netherlands] ; Boston, [Mass.] , pp. 135-147. ISBN 9780123869050

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Abstract

The hydroxylase component of soluble methane monooxygenase (sMMO), which is the site of oxidation of methane and many adventitious substrates of commercial and environmental interest, has proved challenging to manipulate genetically because of difficulties with obtaining functional expression in Escherichia coil. Here, we describe methods that allow site-directed mutagenesis of the hydroxylase-encoding genes and subsequent production of mutant proteins in a modified strain of a methane-oxidizing bacterium, using methane as the carbon and energy source. Mutagenesis and other genetic manipulations are performed in E. coli via standard methods and then, a shuttle plasmid is used to transfer the mutant genes via conjugation to a strain of Methylosinus trichosporium in which the chromosomal copy of the sMMO operon has been partially deleted. Expression is directed by the natural sMMO promoter at high cell density under appropriate culture conditions. The system is not restricted to active mutants of sMMO because Ms. trichosporium can grow on methane using the membrane-associated particulate methane monooxygenase (pMMO) even when it has no active sMMO.

Item Type:	Book Item
Alternative Title:
Divisions:	Faculty of Science > Life Sciences (2010- )
Series Name:	Methods in enzymology
Publisher:	Elsevier/Academic Press
Place of Publication:	Amsterdam, [Netherlands] ; Boston, [Mass.]
ISBN:	9780123869050
Book Title:	Methods in methane metabolism. Part B , Methanotrophy
Editor:	Rosenzweig, A. C. and Ragsdale, S. W.
Date:	2011
Volume:	Vol.495
Number of Pages:	309
Page Range:	pp. 135-147
Status:	Peer Reviewed
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/41534

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