Cloning and expression of a Pseudomonas 3 alpha-hydroxysteroid dehydrogenase-encoding gene in Escherichia coli

A bacterial strain, B-0831, which produced 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) was isolated and identified as belonging to the genus, Pseudomonas. Molecular weights of the purified 3 alpha HSD, determined by SDS-PAGE and by chromatography on Sephacryl S-200, were about 25 and 50 kDa, respectively. A genomic library of Pseudomonas sp. B-0831, prepared in the plasmid vector pACYC184, was screened with probes based on the amino acid (aa) sequence of the protein to obtain the plasmid, p3 alpha HSD1, identified by hybridization with the probes, that contained a 2.4-kb insert from Pseudomonas DNA. When the 1.4-kb SphI fragment of p3 alpha HSD1 was inserted into the vector, pUC118, and introduced into Escherichia coli DH1 under the control of lacZ promoter in the vector, the transformants produced 200-fold more 3 alpha HSD intracellularly than Pseudomonas sp. B-0831. Sequence analysis of the 3 alpha HSD gene revealed that an ORF encoding 3 alpha HSD consists of 254 aa, with a calculated M(r) of 25,761, suggesting that the enzyme consists of homodimer subunits.