Mutagenesis of soluble methane monooxygenase

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 coli. 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.