Cloning and expression in E. coli of the HOPDA hydrolase gene from Pseudomonas sp. P20

Pseudomonas sp. P20 is a natural isolate which is capable of degrading biphenyl and 4-chlorobiphenyl. From a clone of pCK1022 harboring pcbCD genes of Pseudomonas sp. P20, a pcbD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase was subcloned in Escherichia coli XL1-Blue by using pBluescript SK(+) vector. The 2.8-kb HindIII fragment harboring the pcbD gene cloned in pCK1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 carrying pCK1024 degraded HOPDA to benzoate and 2-hydroxypenta-2,4-dienoate by HOPDA hydrolase encoded by pcbD gene as effectively as E. coli CK 1022 harboring pcbCD genes.