Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b

An homologous expression system has been developed for soluble methane mono oxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mut ants were previously obtained after marker-exchange mutagenesis, by the ins ertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO op eron. Complementation of the sMMO-minus genotype was achieved by conjugatio n with broad-host-range plasmids containing the native promoter and sMMO op eron from Ms. trichosporium OB3b (pVK100Sc and pHIM2). In wild-type methano trophs, copper ions present in the growth medium at concentrations greater than 0.25 mu M inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensi tivity to copper, since expression of sMMO occurred at copper sulphate conc entrations of 7.5 mu M. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibite d by copper ions in vitro. This phenomenon could have arisen due to the inc reased number of sMMO gene copies (derived from pVK100Sc) in the cell. Tran sconjugants obtained from conjugations with pHM2 expressed sMMO at copper c oncentrations of 0-2.5 mu M only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher th an 2.5 mu M. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. T his is the first report of homologous sMMO expression in a methanotroph wit h enzyme activities that are comparable to the activity reported in wild-ty pe strains. This expression system will be useful for site-directed mutagen esis of active-site residues of sMMO from Ms. trichosporium OB3b.