Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b

摘要

Summary: An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMM minus mutants were previously obtained after marker-exchange mutagenesis by the insertion of a kanamycin-resistance cassette into the mmoX gene of th sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0·25 μM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7·5 μM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2·5 μ only and sMMO activity was not restored by the addition of purified reductas component at copper concentrations higher than 2·5 μM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.