Tuning biphenyl dioxygenase for extended substrate specificity

Highly substituted polychlorinated biphenyls (PCBs) are known to be very re sistant to aerobic biodegradation, particularly the initial attack by biphe nyl dioxygenase. Functional evolution of the substrate specificity of biphe nyl dioxygenase was demonstrated by DNA shuffling and staggered extension p rocess (StEP) of the bphA gene coding for the large subunit of biphenyl dio xygenase. Several variants with an extended substrate range for PCBs were s elected. In contrast to the parental biphenyl dioxygenases from Burkholderi a cepacia LB400 and Pseudomonas pseudoalcaligenes KF707, which preferential ly recognize either ortho-(LB400) or para-(KF707) substituted PCBs, several variants degraded both congeners to about the same extent. These variants also exhibited superior degradation capabilities toward several tetra- and pentachlorinated PCBs as well as commercial PCB mixtures, such as Aroclor 1 242 or Aroclor 1254. Sequence analysis confirmed that most variants contain ed at least four to six template switches. All desired variants contained t he Thr335Ala and Phe336lle substitutions confirming the importance of this critical region in substrate specificity. These results suggest that the bl ock-exchange nature of gene shuffling between a diverse class of dioxygenas es may be the most useful approach for breeding novel dioxygenases for PCB degradation in the desired direction. (C) 1999 John Wiley & Sons, Inc.