Biphenyl dioxygenases (BP Dox) from different organisms, which are involved in the initial oxygenation and subsequent degradation of polychlorinated biphenyls (PCB), are similar in structure but have different functions. The large subunit of BP Dox, encoded by the bphA1 gene, is crucial for substrate selectivity. Using the process of DNA shuffling, we randomly recombined the bphA1 genes of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 and selected for genes that expressed proteins with altered function. Upon expression in Escherichia coli, some of these evolved genes exhibited enhanced degradation capacity, not only for PCB and related biphenyl compounds, but for single aromatic hydrocarbons such as benzene and toluene, which are poor substrates for the original BP Dox.

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.Biphenyl-utilizing bacteria have been extensively studied in terms of the degradation of polychlorinated biphenyls (PCB), which have been recognized as some of the most significant environmental pollutants (7). These PCB-degrading bacteria exhibit substantial differences in the range of degradation ability and in congener selectivity for PCB. The biphenyl dioxygenases (Bph Dox) are involved in the initial oxygenation of biphenyl and thereby the cometabolic degradation of PCB (10). The Bph Dox of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 exhibit distinct differences in the substrate range for PCB (6, 9), although these two Bph Dox share over 95% identity in their amino acid sequences (5, 22). These Bph Dox are multicomponent enzymes encoded by four genes, bphA1A2A3A4, where bphA1 encodes a large subunit (BphA1) of the terminal dioxygenase (an iron-sulfur protein), bphA2 encodes a small subunit (BphA2) of the terminal dioxygenase, bphA3 encodes the ferredoxin (BphA3), and bphA4 encodes the ferredoxin reductase (BphA4) (5, 22). BphA1 contains a [2Fe-2S] Rieske center which is involved in electron transfer from the ferredoxin component to a mononuclear Fe 2ϩ , which is believed to activate molecular oxygen (1,13,17). Among these four subunits, BphA1 is crucially responsible for the recognition and binding of the substrates and thereby for substrate specificity (6, 10, 15). Previously, we constructed various bphA1 variants by using DNA shuffling between the KF707 and LB400 bphA1 genes (16). Some of the evolved Bph Dox thus obtained exhibited enhanced abilities to degrade PCB and some biphenyl-related compounds. Further screening of these clones allowed us to obtain evolved Bph Dox which exhibit extremely enhanced abilities to degrade benzene, toluene, and alkylbenzenes, such as ethylbenzene, isopropylbenzene, and butylbenzene.A library of evolved bphA1 genes was created by DNA shuffling between the bphA1 genes of strains KF707 and LB400 as previously described (16). The shuffled bphA1 genes were digested with SacI and BglII, inserted just upstream of bphA2A3A4BC in pJHF18⌬MluI, and transformed into Escherichia coli XL1-Blue. The clones grown on Luria-Bertani ag...

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