Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme

Abstract

Dehydroquinate synthase has been purified 9000-fold from Escherichia coli K-12 (strain MM294). The synthase is encoded by the aroB gene, which is carried by plasmid pLC29-47 from the Carbon-Clarke library. Construction of an appropriate host bearing pLC29-47 results in a strain that produces 20 times more enzyme than strain MM294. Subcloning of the aroB gene behind a tac promoter results in E. coli transformants that produce 1000 times more enzyme than MM294: the synthase constitutes 5% of the soluble protein of the cell. A laborious isolation from 50 g of wild-type E. coli cells yields 80 micrograms of impure enzyme, whereas 50 g of cells containing the subcloned gene yields 150 mg of homogeneous enzyme in a two-column purification. Dehydroquinate synthase is a monomeric protein of Mr 40 000-44 000. The chromosomal enzyme from E. coli K-12, the cloned enzyme encoded by the plasmid pLC29-47, and the subcloned inducible enzyme encoded by pJB14 all comigrate on polyacrylamide gel electrophoresis under denaturing conditions.