Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein.
The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.
van den Hombergh
J P
JP
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, U.K.
Moore
J D
JD
Charles
I G
IG
Hawkins
A R
AR
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008190:Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009876:Operon; D019761:Phosphorus-Oxygen Lyases; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014166:Transferases
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C019728:arom enzyme; D000429:Alcohol Oxidoreductases; D014166:Transferases; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases