Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Abstract

A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.