Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates

Abstract

Soluble methane monooxygenase (sMMO) from methane-oxidising bacteria can oxygenate more than 100 hydrocarbons and is one of the most catalytically versatile biological oxidation catalysts. Expression of recombinant sMMO has to date not been achieved in Escherichia coli and so an alternative expression system must be used to manipulate it genetically. Here we report substantial improvements to the previously described system for mutagenesis of sMMO and expression of recombinant enzymes in a methanotroph (Methylosinus trichosporium OB3b) expression system. This system has been utilised to make a number of new mutants and to engineer sMMO to increase its catalytic precision with a specific substrate whilst increasing activity by up to 6-fold. These results are the first 'proof-of-principle' experiments illustrating the feasibility of developing sMMO-derived catalysts for diverse applications.

Keywords: biocatalysis; hydrocarbon oxidation; methane; monooxygenase; protein engineering.

Figure 1.

(a) Components of sMMO illustrating how the recombinant (and wild-type) whole-cell biocatalysts allow oxygenation of methane and other substrates (X converted to XO), driven by O₂ and electrons supplied from externally supplied formate via formate dehydrogenase. The three subunits of the hydroxylase component of sMMO are shown in grey. The reductase component (MmoC—supplies electrons from NADH) is shown in turquoise and protein B (MmoB—also needed for full activity) in dark blue. (b) α Subunit of the M. trichosporium sMMO hydroxylase (constructed using crystallographic data from Elango et al. (1997)) showing the active centre (iron atoms as pink spheres; iron ligands in pink; residues lining the hydrophobic pocket in green) and representative residues of each of the three ionic networks (brown) around the proposed pathway of ingress of substrates into the active site. (c) Expanded view of the active centre showing the mutated residues F192 and I217, together with L110 mutated previously and F188. F188 is spatially close to F192 mentioned in the text.

Figure 2.

Construction of cloning vector pT2ML. The stout arrow within the sMMO-encoding operon indicates the approximate location of the natural promoter that directs expression of the sMMO structural genes at low copper-to-biomass ratio. In order to create the plasmids for expression of the recombinant sMMO variants, the internally deleted ΔmmoX (shaded in dark grey) was replaced by the full-length mutant (or wild-type) mmoX gene fragment via cloning with BamHI and NdeI.