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Sequence and overexpression of the menD gene from Escherichia coli.

Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.

ABSTRACT

The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase. DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein. Possible promoter and ribosome binding sites are present. Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector. This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes.

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