A novel phenanthrene dioxygenase from Nocardioides sp strain KP7: Expression in Escherichia coli

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. T his organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanth rene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was clon ed and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the al pha and beta subunits of the oxygenase component, a ferredoxin, and a ferre doxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously c haracterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene, PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC seque nce showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of th e [2Fe-2S] type of ferredoxin that has been found in most of the reported r ing-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) s equence identity to known reductases. The phdABCD genes were expressed poor ly in Escherichia coli, even when placed under the control of strong promot ers. The introduction of a Shine-Dalgarno sequence upstream of each initiat ion codon of the phdABCD genes improved their expression in E. coli. E. col i cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activ ity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading a ctivity which was significantly less than that in cells carrying the phdABC D genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The geneti c organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthr ene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromati c ring-hydroxylating dioxygenases.