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Journal of Bacteriology, September 2001, p. 5441-5444, Vol. 183, No. 18
Department of Biosciences and Biotechnology,
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received 1 May 2001/Accepted 27 June 2001
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 Fe2+, 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 The degradative activities of the 80 positive clones toward
diphenylmethane and dibenzofuran are presented in Fig.
1. First, it is noted that
Escherichia coli(pSHF707) expressing the original BphA1
(KF707 enzyme) and E. coli(pSHF400) expressing the original LB400 BphA1 (LB400 enzyme) exhibited major differences in the formation
of the meta ring cleavage yellow products for these two
compounds. The yellow compound 2-hydroxy-6-oxo-6-benzylhexa-2,4-dienoic acid was produced from diphenylmethane by the KF707 enzyme but not by
the LB400 enzyme. In contrast, the yellow compound
2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoic acid was produced
from dibenzofuran by the LB400 enzyme but not by the KF707 enzyme. The
formation of yellow meta ring-cleavage products from
diphenylmethane and dibenzofuran were monitored with the supernatants
at the corresponding absorption maxima. The amount of yellow
meta ring cleavage product from diphenylmethane was 4.64 nmol/mg of protein/min by E. coli expressing the original KF707 Bph Dox, and that from dibenzofuran by the original LB400 Bph Dox
was 3.16 nmol/mg of protein/min. The 80 positive clones which exhibited
both diphenylmethane and dibenzofuran degradation activities were
screened, and the relative abilities to degrade these two compounds
were determined, where the activities of E. coli expressing
the above original Bph Dox were set to 100 as the basal activities.
Among these, we selected five clones, E. coli carrying
pSHF1030, pSHF1045, pSHF1049, pSHF1071, and pSHF1072, which produced
indigo on Luria-Bertani agar plates, because it is known that
oxygenases capable of metabolizing monocyclic aromatic compounds such
as toluene, phenol, and styrene transform indole to indigo (3, 4,
11, 18). As expected, these clones exhibited enhanced abilities
to degrade ethylbenzene, isopropylbenzene, and butylbenzene (Fig.
2). These clones produced almost the same amount of Bph Dox, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). In particular, E. coli(pSHF1072) was interesting because this clone exhibited wide
and enhanced capabilities to degrade not only the alkylbenzenes but
also benzene and toluene, compounds scarcely attacked by the original
Bph Dox. The amounts of yellow compounds produced from ethylbenzene,
isopropylbenzene, and butylbenzene by this clone were two to seven
times those produced by E. coli(pSHF707) and E. coli(pSHF400) expressing the respective original Bph Dox. Sequence
analyses of the evolved BphA1 that acquired the higher activities for
the alkylbenzenes gained the same two amino acids from the LB400
enzyme, i.e., His255Gln and Val258Ile (KF707 numbering) (Fig.
3). These results suggest that alterations of His-255 and Val-258 in the KF707 enzyme are important for the enhancement of substrate specificity toward monocyclic aromatic
hydrocarbons. In order to investigate this point, site-directed mutagenesis was applied to pSHF1072, in which two or three amino acids
of the four substituted amino acids from the LB400 enzyme were changed
to those of the KF707 enzyme.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5441-5444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Directed Evolution of Biphenyl Dioxygenase:
Emergence of Enhanced Degradation Capacity for Benzene, Toluene,
and Alkylbenzenes
ABSTRACT
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MluI, and transformed into Escherichia
coli XL1-Blue. The clones grown on Luria-Bertani agar
plates containing ampicillin at 50 µg/ml and 0.1 mM
isopropyl-
-D-thiogalactopyranoside
were screened for the ability to produce yellow pigment from biphenyl
(8). Eighty positive clones thus selected were then
analyzed for the ability to degrade biphenyl-related compounds and
monocyclic aromatic hydrocarbons. The production of yellow
meta ring cleavage products from various aromatic
compounds was monitored with the supernatants at the following
absorbance wavelengths: biphenyl, 434 nm; diphenylmethane, 395 nm;
dibenzofuran, 465 nm; benzene, 388 nm; toluene, 375 nm; ethylbenzene,
319 nm; isopropylbenzene, 321 nm; butylbenzene, 323 nm.
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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.
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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.
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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.
Bph Dox from pSDF2073 and pSDF2076 possess the amino acids Q255 and I258 and the amino acid I258, respectively, from the LB400 enzyme. E. coli expressing these enzymes exhibited slightly greater activities against all of the substrates tested than did the KF707 enzyme but much less activity than the pSHF1072 enzyme. However, these enzymes retained relatively greater activity against toluene. Bph Dox from pSDF2075, in which only Gln-255 was derived from the LB400 enzyme, exhibited almost the same activity for biphenyl and butylbenzene as did the pSHF1072 enzyme but much lower activities against benzene and toluene. Bph Dox from pSDF2074 exhibited activities similar to those of the KF707 enzyme, in which three amino acids at positions 255, 258, and 277 were the reverse of those of the KF707 enzyme.
It was previously shown that the replacement of Thr-376 with Asn led to the acquisition of 3,4-dioxygenase activity for 2,5,4'-trichlorobiphenyl and 2,5,2',5'-tetrachlorobiphenyl (15, 20). Furthermore, a change to Val at the same position produced the novel abilities to degrade dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene (21). These results indicated that the amino acid at position 376 in BphA1 plays a very important role in determining substrate selectivity. Therefore, a variant, pSHF1072N, in which Thr-376 in the pSHF1072 enzyme was changed to Asn, was created. The Bph Dox from pSHF1072N exhibited almost the same degradation ability as the original Bph Dox from pSHF707 and pSHF400 for the substrates tested (Fig. 2). The Bph Dox from pSHF34 (20) and pSHF1046, in which Thr-376 in the KF707 enzyme was replaced with Asn and Asn-376 in the LB400 enzyme was replaced with Thr, respectively, hardly attacked benzene, toluene, and ethylbenzene (data not shown). These results suggest that even one amino acid change at position 376 in BphA1 significantly affected the ability to degrade monocyclic aromatic hydrocarbons and that the combination of Gln-255, Ile-258, Ala-268, Tyr-277, and Thr-376 is important for the enhanced degradation of benzene and toluene, as seen in pSHF1072.
It has been reported that the toluene dioxygenase of Pseudomonas putida F1 shows a wide range of substrate specificities for various aromatic compounds (2, 12, 23). In this study, we found that the toluene dioxygenase from F1 exhibited high oxygenation activities toward benzene (316% of pSHF1072 Bph Dox) and toluene (142%) but relatively low activities for ethylbenzene (29%), butylbenzene (25%), and isopropylbenzene (32%), of which the latter three compounds have bulky side groups. In naphthalene dioxygenase, replacement of Thr-351, corresponding to Thr-376 in KF707 BphA1, with Arg had a large effect on product formation from phenanthrene (19). Although the three-dimensional structure of Bph Dox is not available yet, that of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4, whose structure is similar to that of Bph Dox, was solved by X-ray analysis (14). Based on the structural information from the naphthalene dioxygenase, we tried to analyze the possible structure of KF707 BphA1 (data not shown). The results indicate that the four amino acids in pSHF1072 BphA1 are situated surrounding a mononuclear iron center which is supposed to be an active site. The flexibility of amino acids near the active site may lead to the relaxation of substrate binding and allow expansion of the abilities of the pSHF1072 enzyme to degrade a variety of aromatic hydrocarbons.
Thus, more appropriate combinations of amino acids involved in substrate recognition will allow us to evolve new and novel enzymes with much wider and enhanced oxygenation capacities. DNA shuffling is an effective approach by which to get such evolved enzymes.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biosciences and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Phone: 81-92-642-2849. Fax: 81-92-642-2849. E-mail: kfurukaw{at}agr.kyushu-u.ac.jp.
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Journal of Bacteriology, September 2001, p. 5441-5444, Vol. 183, No. 18
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.18.5441-5444.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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