Cloning, expression, and sequence analysis of a lignostilbene-alpha,beta-dioxygenase gene from Pseudomonas paucimobilis TMY1009.

From the genomic library of Pseudomonas paucimobilis TMY1009 constructed with the cosmid pHC79, an 8-kb BamHI-KpnI fragment encoding lignostilbene-alpha,beta-dioxygenase (LSD) was cloned into pUC19 (designated pKSN2510). E. coli JM109 having pKSN2510 produced a small amount of LSD only when the lac promoter was induced by isopropyl-beta-D-thio-galactopyranoside (IPTG). The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUC119 in opposite directions (designated pKHN2560 and pKHN2590). The LSD gene on pKHN2590 was expressed in E. coli MV1184 using the lac promoter. LSD produced by E. coli MV1184 transformed with pKHN2590 (cloned LSD) was purified and found to be identical with LSD-I, which was the major isozyme produced by P. paucimobilis TMY1009. Cloned 1.9-kb nucleotide was sequenced and one open reading frame composed to 486 codons was found.