Heterologous expression and site-directed mutagenesis of soluble methane monooxygenase
The purpose of this investigation was to study the heterologous expression of soluble methane monooxygenase (sMMO) genes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. Using the T7-RNA polymerase expression system, the entire sMMO operon and subclones (constructed using the polymerase chain reaction) were over-expressed in E. coli. Results obtained using the Me. capsulatus (Bath) sMMO operon confirmed previous reports (C. West, G. P. C. Salmond, H. Dalton and 1. C. Murrell (1992). J Gen. Microbial. 138, 1301-1307) that functional expression of protein B and the reductase occurred but the hydroxylase was inactive. Similar results were obtained by expressing the sMMO operon of Ms. trichosporium OB3b in E. coli, using plasmids previously described (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbiol. 60, 2473-2482). Protein B, the reductase and orfY were over-expressed and purified from E. coli using glutathione-Stransferase fusion proteins and affinity chromatography. The expression of sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Pseudomonas putida. A previous report (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbial. 60, 2473-2482) had suggested that functional expression of sMMO from Ms. trichosporium OB3b was achieved in P. putida Fl. Attempts to repeat this work proved that protein B and the reductase were functionally expressed, but the hydroxylase was inactive. Similar results were obtained for the heterologous expression of the sMMO operon from Mc. capsulatus (Bath) in P. putida. Methanotrophs were used for the heterologous expression of sMMO via two strategies. (1) The expression of sMMO from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Methylomonas album B08 and Methylocystis parvus OBBP. These are methanotrophs that do not express sMMO, but express particulate MMO (pMMO) only, to utilise methane as a sole carbon and energy source. Functional expression of the sMMO operon of Ms. trichosporium OB3b was achieved in Mm. album BG8, however, recombinant sMMO enzyme activity was poor and problems were encountered with the growth of the sMMO positive transconjugant methanotrophs. (2) sMMO-minus marker exchange mutants of Ms. trichosporium OB3b (H. Martin and 1. C. Murrell (1995). FEMS Microbiol. Letts. 127, 243-248) were complemented with plasmid encoded genes and functional sMMO expression was obtained. Southern hybridisation analysis revealed that the plasmid DNA had integrated into the chromosome of the Ms. trichosporium OB3b sMMO-minus mutant via a single homologous recombination event between the mmoX genes. Protein B from Mc. capsulatus (Bath) is inactivated by proteolysis to give rise to a truncated form designated protein B'. The Met 12-Gly 13 cleavage site was modified by site-directed mutagenesis to Met12-Gln13 which improved the stability of the protein when incubated at room temperature. Only after prolonged incubation was protein B' formed. Recombinant protein B from Ms. trichosporium OB3b also appears to be unstable, and readily degraded when incubated at room temperature. The cleavage of protein B to inactive protein B' may be a general regulatory mechanism that occurs within the cell to regulate sMMO activity.