Cloning and characterization of the genes encoding enzymes for the protocatechuate meta-degradation pathway of Pseudomonas ochraceae NGJ1

The 2-pyrone-4,6-dicarboxylate lactonase gene (proL), the protocatechuate 4,5-dioxygenase alpha and beta subunits genes (proOa and proOb), and the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (proD) were cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1. These genes were in the order proLOaObD on the DNA, and a possible transcription terminator sequence followed. The proL and proD genes were over-expressed in Escherichia coli, and their gene products were purified for identification, while the expression of proOaOb was at a lower level. The protocatechuate meta-degradation operon was reconstituted with the recombinant plasmids and expressed successfully in E. coli.