Expression vectors for the methane-producing archaeon Methanococcus maripaludis
Gardner, Warren Lynwood
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Methanogens are strict anaerobes that use one and two carbon compounds for methanogenesis. They contain many unusual cofactors and enzymes, and many of their proteins are oxygen-sensitive and are present at low concentrations within the cells. An expression system that overexpresses homologous and heterologous proteins would facilitate research on the unique oxygen-sensitive enzymes in these organisms. However, traditional expression systems may lack the cofactors and maturation enzymes necessary for the expression of the active holoenzymes. Therefore, a major goal of this work was to develop expression vectors. To develop a shuttle vector, a series of integrative vectors were first constructed. The integrative vectors were based on the pUC-derivative pMEB.2. pMEB.2 provided the puromycin resistance marker for methanococci, an Escherichia coli origin of replication, and an ampicillin resistance marker for E. coli. A multiple cloning site and the Methanococcus voltae histone promoter (PhmvA ) were added to form the integrative, expression vector pWLG14. To demonstrate the utility of PhmvA , pWLG14 was used to overexpress the genomic copy of the Methanococcus maripaludis acetohydroxyacid synthase. To form an expression shuttle vector suitable for heterologous genes, pWLG14 and the cryptic plasmid pURB500 from M. maripaludis strain were ligated together to form pWLG30. pWLG30 was the first expression shuttle vector for the methanogens. To demonstrate the utility of pWLG30, the E. coli b-galactosidase gene was cloned into pWLG30 to yield pWLG30+lacZ. Upon transformation into M. maripaludis, the recombinant strain expressed ?-galactosidase to the level of 1% of the cellular protein. pWLG30+lacZ also provided a convenient vector for the cloning because it provided a blue/white screen in E. coli. This vector was then used to express the carbon monoxide dehydrogenase (CODH), pyruvate oxidoreductase, monomethylamine methyltransferase, and a subunit of the F420-reducing hydrogenase in M. maripaludis. A transposable element was also used to mutate pWLG30+lacZ to identify regions essential for plasmid replication in M. maripaludis.