Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes

Abstract

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.

FIG. 1

Formation of yellow meta ring cleavage products from diphenylmethane and dibenzofuran by E. coli expressing chimeric biphenyl dioxygenases. The cells were incubated with 0.1 mM substrate at 30°C for 1 h. The formation of yellow compounds was measured at the corresponding absorption maximum. The degradation activities of KF707 Bph Dox and LB400 Bph Dox were used as the basal activities (set to 100) toward diphenylmethane and dibenzofuran, respectively. The activity of KF707 Bph Dox is shown by the larger square on the x axis, and that of LB400 Bph Dox is shown by the larger triangle on the y axis. The closed circles indicate the activities in E. coli expressing the evolved Bph Dox. The relative degradation activities of 80 clones were plotted for the basal activities.

FIG. 2

Formation of meta-cleaved yellow compounds from a variety of aromatic hydrocarbons by E. coli expressing chimeric Bph Dox. Equal amounts of E. coli cells expressing evolved Bph Dox were incubated with the substrate at 30°C for 1 h (biphenyl, isopropylbenzene, butylbenzene, and ethylbenzene) or 8 h (benzene and toluene). The formation of the yellow compounds was measured at the corresponding absorption maximum. The results are shown as average values ± the standard deviation of three independent experiments. O.D., optical density.

FIG. 3

Sequence analyses of the resultant shuffled bphA1 genes. Twenty amino acids that differ between KF707 BphA1 and LB400 BphA1 are shown with KF707 numbering at the top. The dash indicates an amino acid lacking in KF707 relative to the LB400 sequence. The pSDF series plasmids were constructed by site-directed mutagenesis.