Cloning and expression of the Thiobacillus ferrooxidans 3-isopropylmalate dehydrogenase gene in Escherichia coli

The 3-isopropylmalate (3-IPM) dehydrogenase [EC 1.1.1.85] gene leuB of an acidophilic autotroph Thiobacillus ferrooxidans was cloned and expressed in Escherichia coli. Recombinant plasmids pTFL1 and pTFL2, carrying a 6.7-kb PstI fragment in the opposite orientation, conferred the same level of 3-IPM dehydrogenase activity on an E. coli leuB mutant. Restriction endonuclease mapping and deletion analysis indicated that 3-IPM dehydrogenase gene was on a 3.1-kb PstI-NruI fragment. The expression of the 3-IPM dehydrogenase gene of T. ferrooxidans was repressed by the addition of leucine in the culture medium of E. coli carrying the plasmid pTFL1. These results indicate that the 6.7-kb fragment contains the leuB structural gene and its regulatory region.