Expression vectors for Methanococcus maripaludis: Overexpression of acetohydroxyacid synthase and beta-galactosidase

A series of integrative and shuttle expression vectors was developed for us e in it Methanococcus maripaludis. The integrative expression vectors conta ined the il Methanococcus voltae histone promoter and multiple cloning site s designed for efficient cloning of DNA. Upon transformation, they can be u sed to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than th at of the wild type. An expression shuttle vector, based on the cryptic pla smid pURB500 and the components of the integrative vector, was also develop ed for the expression of heterologous genes in M. maripaludis. The beta-gal actosidase gene from Escherichia coli was expressed to similar to 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (pps A) were sequenced from a M. maripaludis genomic library.