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.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...

Steady-State Kinetic Characterization of Evolved Biphenyl Dioxygenase, Which Acquired Novel Degradation Ability for Benzene and Toluene

“…In particular, it is noteworthy that an Escherichia coli clone carrying pSHF1072 plasmid exhibits wide and enhanced capabilities to degrade not only alkylbenzenes but also benzene and toluene, compounds that are rarely attacked by wild-type KF707 Bph Dox. 8) Sequence analysis of the pSHF1072 bphA1 gene revealed that four amino acids (His255Gln, y To whom correspondence should be addressed. Tel/Fax: +81-29-861-6733; E-mail: suenaga-hikaru@aist.go.jp * Present address: Fuji Gotemba Research Labs, Chugai Pharmaceutical Co., Ltd., Gotemba 412-8513, Japan Abbreviations: Bph Dox, biphenyl dioxygenase; PCB, polychlorinated biphenyl; MOPS, 4-morpholinepropanesulfonic acid; MEG, 50 mM MOPS buffer pH 7.0, containing 5% ethanol and 5% glycerol Val258Ile, Gly268Ala, and Phe277Tyr) were changed from KF707 BphA1 to those in the LB400 BphA1.…”

“…E. coli strain JM109 carrying pHSF1072 8) was grown at 37 C in 1 liter of Luria-Bertani medium containing 50 mg/ml ampicillin and 0.1 mM isopropyl--D-galactopyranoside (IPTG) from a 1% (v/v) seeding of an overnight preculture. After 12 h of incubation, the E. coli cells were harvested and suspended in a 50 mM MOPS buffer (pH 7.0) containing 5% ethanol and 5% glycerol (MEG).…”

Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes

“…Mutants of toluene dioxygenase were generated by random and saturation mutagenesis that exhibited higher activity towards toluene and 4-picoline (Sakamoto et al, 2001). Mutants of biphenyl dioxygenases with enhanced substrate specificity and changed regioselectivity were obtained by random and site-directed mutagenesis and gene shuffling (Brühlmann & Chen, 1999;Erickson & Mondello, 1993;Suenaga et al, 2001). In this study, rational design was used in order to change the product spectra of TecA.…”

Biodegradation of Cypermethrin by Two Isolates of Pseudomonas aeruginosa

“…Because, the native strains grow very good and have advanced adaptability in particular geographical region [10]. native microorganisms are highly adaptable and have the capability to degrade the recalcitrant compounds through evolution of new genes, which encode enzymes that can use these compounds as their primary substrates [11].…”