CLONING, NUCLEOTIDE-SEQUENCE, AND OVEREXPRESSION OF THE GENE CODING FOR DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-PUTIDA BIOTYPE-B

The structural gene coding for the Delta(5)-3-ketosteroid isomerase (K SI) of Pseudomonas putida biotype B has been cloned, and its entire nu cleotide sequence has been determined by a dideoxynucleotide chain ter mination method. A 2.1-kb DNA fragment containing the ksi gene was clo ned from a P. putida biotype B genomic library in lambda gt11. The ope n reading frame of ksi encodes 393 nucleotides, and the amino acid seq uence deduced from the nucleotide sequence agrees with the directly de termined amino acid sequence (K. Linden and W. B. Benisek, J. Biol. Ch em. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21 (DE3) transformed with the pKK-KSI plasmid containing the ksi gene exp ressed a high level of isomerase activity when induced by isopropyl-be ta-D thiogalactopyranoside. KSI was purified to homogeneity by a simpl e and rapid procedure utilizing fractional precipitation and an affini ty column of deoxycholate-ethylenediamine-agaro se as a maj or chromat ographic step. The molecular weight of KSI was 14,535 (calculated, 14, 536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mu mol min(-1) mg(-1)) and a K-m ( 60 mu M) which are close to those of KSI originally obtained from P. p utida biotype B.