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J Biol Chem, Vol. 275, Issue 14, 10085-10092, April 7, 2000
From the Xylene monooxygenase of Pseudomonas
putida mt-2 catalyzes the methylgroup hydroxylation of toluene
and xylenes. To investigate the potential of xylene monooxygenase to
catalyze multistep oxidations of one methyl group, we tested
recombinant Escherichia coli expressing the monooxygenase
genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans
Gpo1. Expression of xylene monooxygenase genes could efficiently be
controlled by n-octane and dicyclopropylketone. Xylene
monooxygenase was found to catalyze the oxygenation of toluene,
pseudocumene, the corresponding alcohols, and the corresponding
aldehydes. For all three transformations 18O incorporation
provided stong evidence for a monooxygenation type of reaction, with
gem-diols as the most likely reaction intermediates during
the oxygenation of benzyl alcohols to benzaldehydes. To investigate the
role of benzyl alcohol dehydrogenase (XylB) in the formation of
benzaldehydes, xylB was cloned behind and expressed in
concert with xylMA. In comparison to E. coli
expressing only xylMA, the presence of xylB
lowered product formation rates and resulted in back formation of
benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB
may prevent the formation of high concentrations of the particularly
reactive benzaldehydes. In the case of high fluxes through the
degradation pathways and low aldehyde concentrations, XylB may
contribute to benzaldehyde formation via the energetically favorable
dehydrogenation of benzyl alcohols. The results presented here
characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes.
Xylene monooxygenase
(XMO),1 encoded by the
plasmid pWW0 of Pseudomonas putida mt-2, is a key enzyme
system in the degradation of toluene and xylenes. XMO is a member of
the alkyl-group hydroxylase family and hydroxylates the methyl side
chain of the aromatic ring. This is the first step toward the
production of a carboxylic acid (upper degradation pathway for xylenes)
(Fig. 1), which is then transformed to
substrates of the Krebs cycle through the meta cleavage
pathway. The upper pathway enzymes are encoded by the upper operon
(Fig. 1), whereas the meta cleavage pathway is encoded by
the meta operon (1-5). XMO consists of two polypeptide subunits,
encoded by xylM and xylA (2, 6). XylA, the
NADH:acceptor reductase component, was characterized as electron
transport protein transferring reducing equivalents from NADH to XylM
(7). XylM, the hydroxylase component, is located in the membrane, and
its activity depends on phospholipids and ferrous ion with a pH optimum of 7 (8, 9). The XylM amino acid sequence has a 25% homology with
AlkB, the hydroxylase component of alkane hydroxylase of Pseudomonas oleovorans GPo1 (6). Alkane hydroxylase is the first enzyme in the degradation of medium chain length alkanes through
a set of enzymes encoded on two alk gene clusters on the catabolic OCT plasmid (10-13). Sequence analyses indicated that at
least 11 nonheme integral membrane enzymes, including AlkB and XylM,
contain a highly conserved 8-histidine motif (14, 15). This motif was
shown to be essential for catalytic activity in rat stearoyl-CoA
desaturase (14). Based on studies of AlkB, the integral membrane
enzymes of this family were proposed to contain diiron clusters as
O2-activating sites, for which the 8-histidine motifs
provide ligands (15).
The second enzyme in the upper pathway is benzyl alcohol dehydrogenase
(BADH), a homodimeric member of the zinc-containing long chain alcohol
dehydrogenase family (16-18). This enzyme is encoded by the
xylB gene. The third enzyme in the upper pathway, benzaldehyde dehydrogenase (BZDH), is also a homodimer and is encoded
by the xylC gene (16, 17).
Using Escherichia coli recombinants, XMO was shown to
oxidize not only toluene and xylenes but also m- and
p-ethyl-, methoxy-, nitro-, and chlorosubstituted toluenes,
as well as m-bromosubstituted toluene to corresponding
benzyl alcohol derivatives (19, 20). Styrene is transformed into
S-styrene oxide with an enantiomeric excess of 95% (20, 21). In
addition, XMO was observed to catalyze the second step in the upper
pathway, the oxidation of benzyl alcohols to corresponding aldehydes
in vivo (2, 22). Even the conversion of benzaldehyde to
benzoate, the third step in the upper pathway, could be observed, but
was attributed to E. coli enzymes (2). Further studies
performed in vitro with partially purified XylMA showed no
activity toward benzyl alcohol (9). The reasons for this discrepancy
remain unclear.
To investigate the ability of E. coli recombinants
expressing XylMA to catalyze the multistep oxidation of one methyl
group of toluene and xylenes in more detail, we used a recently
developed expression system, expressing XMO genes via the
alk regulatory system of P. oleovorans GPo1 (21).
Expression of the first of the two previously mentioned alk
gene clusters is under control of alkBp, the alk
promoter, and is initiated in the presence of the functional regulatory
protein AlkS, which is encoded on the second alk gene
cluster, and alkanes or other inducers such as dicyclopropylketone
(DCPK) (8, 23, 24). In the present study we show that XMO expressed via
the alk regulatory system catalyzes the transformation of
toluene and pseudocumene to the corresponding alcohols, aldehydes, and
acids by consecutive monooxygenation reactions. Furthermore, we
investigated the kinetics of these reactions in the absence and
presence of BADH and show that the presence of BADH results in back
formation of benzyl alcohol from benzaldehyde, thus lowering net
product formation rates. The thermodynamics and the physiological role
of the BADH-catalyzed reaction and the involvement of a
gem-diol intermediate in the XMO-catalyzed alcohol
oxygenation are discussed.
Bacterial Strains and Plasmids--
Strains and plasmids used
are listed in Table I. To test the
enzymatic activities of XylMA containing recombinants of E. coli JM101, an E. coli K-12 derivative, we used pBR322
derived expression vectors equipped with the alk regulatory
system.
Media, Growth Conditions, and Materials--
Bacteria were grown
either on Luria-Bertani broth (Difco, Detroit, MI) or on M9 minimal
medium (25) containing a 3-fold concentration of phosphate salts (M9*)
and 0.5% (w/v) glucose as single carbon source. When necessary,
cultures were supplemented with kanamycin (final concentration, 50 mg/liter), ampicillin (100 mg/liter), chloramphenicol (30 mg/liter),
thiamine (0.001% w/v), 1 mM indole, or 0.5 mM
isopropyl-
Restriction and DNA modification enzymes were obtained from Roche
Molecular Biochemicals, New England Biolabs (Schwalbach, Germany), Life
Technologies, Inc., Angewandte Gentechnologie Systeme (Heidelberg,
Germany), or Promega (Zurich, Switzerland). The QIAprep Spin Miniprep
Kit of Qiagen (Basel, Switzerland) was used to obtain small scale
plasmid DNA preparations following the supplier's protocol. Chemicals
were obtained from Fluka (Buchs, Switzerland) (toluene, >99.5%;
benzyl alcohol, >99%; benzaldehyde, >99%; benzoic acid, >99.5%;
pseudocumene, ~99%; 3,4-dimethylbenzoic acid, ~97%), Aldrich
(Buchs, Switzerland) (3,4-dimethylbenzyl alcohol, 99%), and Lancaster
(Muehlheim, Germany) (3,4-dimethylbenzaldehyde, 97%).
DNA Manipulation and Constructs--
Standard techniques were
used for restriction analysis, cloning, and agarose gel electrophoresis
(25). Construction of pSPZ3 (Fig. 2), a
pBR322 derivative, is described elsewhere (21). To insert the alcohol
dehydrogenase gene xylB into the plasmid pSPZ3 directly
downstream of the xylA gene, the 2.3-kilobase
XhoI/FspI fragment of pCKO4 (26) containing
xylB was first introduced into the XhoI- and
SmaI-digested vector pGEM-7Zf(+) (Promega) to yield pGEMAB.
From this construct the 2.3-kilobase fragment was cut out with
XhoI and BamHI and ligated into the
XhoI- and BamHI-digested plasmid pSPZ3. The
resulting plasmid was called pRMAB (Fig. 2).
As a negative control pRS containing no xyl genes was
constructed. pRMAB was digested with BamHI and
SmaI and treated with Klenow enzyme. After isolation of the
bigger fragment the vector was religated.
Determination of Enzyme Activities in Whole Cell
Assays--
Whole cell biotransformations were carried out with
E. coli JM101 recombinants that were incubated in 40 or 100 ml of medium in the presence of kanamycin. At
A450 of ~0.3, cells were induced by the
addition of 0.05% (v/v) DCPK or 0.1% (v/v) n-octane
(10
One unit is defined as the activity that produces 1 µmol of total
products in 1 min. Specific activity was expressed as activity/g of CDW
(unit g Product Formation as a Function of Time--
Cells were grown,
induced, collected, resuspended, and incubated with substrate as
described for the whole cell activity assays. To follow product
formation over time cell aliquots were incubated with the same
substrate for different time periods (5, 10, 20, 30, 40, and 80 min).
The reactions were stopped as described for the whole cell activity
assays and analyzed.
Analysis of Metabolites--
For high performance liquid
chromatography (HPLC) analysis cells were removed by centrifugation
(7800 × g for 8 min), and the supernatants were
analyzed. For the separation of benzyl alcohol, benzaldehyde, and
benzoic acid, we used a Nucleosil C18 column (pore size, 100 Å;
particle size, 5 µm; inner diameter, 25 cm × 4 mm)
(Macherey-Nagel, Oensingen, Switzerland) with a mobile phase of 69.9%
H2O, 30% acetonitrile, 0.1% H3PO4
at a flow rate of 0.7 ml/min. For the separation of 3,4-dimethylbenzyl
alcohol, 3,4-dimethylbenzaldehyde, and 3,4-dimethylbenzoic acid we used the same column at the same flow rate with a mobile phase of 64.9% H2O, 35% acetonitrile-0.1% H3PO4.
Detection was done using UV light at 210 nm.
For gas chromatography (GC) and gas chromatography-mass spectrometry
(GC-MS) analysis an equal volume of ice-cold ether containing 0.1 mM dodecane as an internal standard was added to samples. After addition of saturating amounts of sodium chloride, the water phase was extracted by vigorous shaking for 5 min at 30 °C, and the
phases were separated by centrifugation. The organic phase was dried
over anhydrous sodium sulfate and analyzed. Gas chromatography was used
to separate toluene/pseudocumene, the respective alcohols, aldehydes,
and acids.
GC analysis was done on a GC (Fisons Instruments) equipped with a fused
silica capillary column OPTIMA-5 (25 m; inner diameter, 0.32 mm; film
thickness, 0.25 µm) of Macherey-Nagel (Oensingen, Switzerland) with
splitless injection and hydrogen as the carrier gas. The following
temperature profile was applied: from 40 to 70 °C at 15 °C/min,
from 70 to 105 °C at 5 °C/min, and from 105 to 240 °C at
20 °C/min. Compounds were detected with a flame ionization detector.
GC-MS analysis was performed on a Fisons MD 800 mass spectrometer and a
gas chromatograph (Fisons Instruments) equipped with a CP-Sil 5CB
column (Chrompack) using split injection (20:1) and helium as the
carrier gas. The temperature program was the same as described for GC
analysis. Substances were identified by comparison of retention times
with those of commercially available standards in both HPLC analysis
and GC analysis and by GC-MS analysis.
18O Incorporation--
E. coli JM101
(pSPZ3) was grown, induced, harvested, and resuspended as described for
the determination of enzyme activities in whole cell assays. 1 ml of
cell suspension was transferred to a Pyrex tube with a total volume of
8.3 ml, which was then sealed by using a butyl rubber stopper and a
screw-on hole cap. Throughout the following steps, the cell suspension
was mixed with a magnetic stirrer. The air in the head space of the
Pyrex tube was removed to a reduced pressure of 20 mbar and replaced with nitrogen three times. The headspace was once again evacuated and
brought to 80% of ambient pressure with nitrogen; following that, the
Pyrex tube was filled with pure oxygen enriched with 18O2 (85 atom % 18O). Following
the final gas exchange, the cell suspension was incubated horizontally
on a rotary shaker at 250 rpm and 30 °C for 15 min to equilibrate
the reaction medium. After equilibration, substrate was added by
injection through the rubber stopper, and the suspension was again
incubated at 250 rpm and 30 °C for different time periods depending
on the substrate (toluene, 3 min; benzyl alcohol, 5 min; benzaldehyde,
90 min; pseudocumene, 5 min; 3,4-dimethylbenzyl alcohol, 20 min; and
3,4-dimethylbenzaldehyde, 20 min). Incubation time was chosen as to
allow a maximal formation of the desired products. Identical
experiments were done with air in the headspace. The reactions were
stopped by placing the samples on ice and immediately adding 40 µl of
a perchloric acid stock solution (10% v/v) immediately followed by
GC-MS analysis as described above.
Growth and Induction Kinetics of E. coli JM101 (pSPZ3)--
XMO
activities were investigated in E. coli JM101
xylMA recombinants. The alk regulatory system,
which can be induced by n-octane or DCPK, was chosen to
control the expression of the XMO genes, xylMA, on the
plasmid pSPZ3 (Fig. 2).
XMO activity was followed after induction with 0.1% (v/v)
n-octane (Fig. 3A)
or 0.05% (v/v) DCPK (results not shown). XMO activity was quickly
induced by both compounds, reaching a constant level of around 115 and
105 units/g of CDW for n-octane and DCPK, respectively,
after 3-3.5 h of induction time. The growth rates of the induced cells
were clearly reduced when compared with that of noninduced cells.
The dependence of XMO activity on inducer concentrations was determined
by growing E. coli JM101 (pSPZ3) to 0.09 g of CDW/liter and inducing the cells with different amounts of n-octane
and DCPK. After further growth for 3.5 h, cell dry weight and XMO activity were determined for each inducer concentration (Fig. 3,
B and C). Very low monooxygenase activities (<6
units/g of CDW) were measured when less than 0.0001% (v/v) octane or
0.001% (v/v) DCPK was added to the culture medium. Maximal induction was observed at DCPK concentrations above 0.005% (v/v), whereas for
n-octane this value was observed at octane volume fractions above 0.001% (v/v).
The cell densities decreased in inducer concentration ranges where
enzyme activities increased to a maximum. For higher concentrations of
n-octane they reached a constant value, whereas higher
concentrations of DCPK provoked a further decrease of cell densities
(Fig. 3, B and C). We therefore checked the
direct influence of different inducer concentrations on the growth of
E. coli JM101 without plasmid. High volume fractions of
n-octane, up to 1% (v/v), had no influence on cell growth.
In contrast, concentrations of DCPK above 0.01% (v/v) led to a
decreased growth rate. At a DCPK concentration of 0.5% (v/v) the CDW
decreased from 0.09 to 0.073 g/liter after 3.5 h of induction,
indicating that high DCPK concentrations are toxic to E. coli host strains.
Oxygenation of Toluene and Derivatives by XMO--
E.
coli JM101 xylMA recombinants were routinely induced by 0.1%
(v/v) n-octane after growth to a cell density of 0.09 g
CDW/liter. After incubation for another 3-3.5 h and concomitant growth
to 0.23-0.27 g of CDW/liter, product formation rates were determined.
XMO was shown to oxygenate toluene to benzyl alcohol, benzyl alcohol to
benzaldehyde, and benzaldehyde to benzoic acid (Table II). For the first two oxygenation
reactions activities as high as 95-100 units/g of CDW were observed,
whereas the oxygenation of benzaldehyde occurred at a lower rate of 10 units/g of CDW. For pseudocumene the results were similar: Pseudocumene
was oxygenated to 3,4-dimethylbenzyl alcohol, 3,4-dimethylbenzyl
alcohol to 3,4-dimethylbenzaldehyde, and 3,4-dimethylbenzaldehyde to
3,4-dimethylbenzoic acid. The hydroxylation of pseudocumene was also
observed to take place at a rate of 100 units/g of CDW, whereas
3,4-dimethylbenzaldehyde was formed more slowly at a rate of 50 units/g
of CDW and oxygenated to 3,4-dimethylbenzoic acid at a clearly higher
rate (55 units/g of CDW) than the oxygenation of benzaldehyde to
benzoic acid. When the acids were added as substrates, no reaction
products and no depletion of acids were detected.
As negative controls the biotransformations were performed with
uninduced E. coli JM101 carrying the plasmid pSPZ3 and with induced E. coli JM101 without plasmid. As an additional
control to exclude any effect of the alk regulatory system
on E. coli, we constructed plasmid pRS, still containing the
alkS gene but lacking the xyl genes. No
conversion products were detected in any of these control experiments
when toluene, pseudocumene, or the corresponding alcohols were added as
substrates (Table II). However, when aldehydes were added as substrates
in these experiments, they were reduced to alcohols at a constant rate
(Table III). The formation of
3,4-dimethylbenzoic acid could also be shown, but at a very low rate of
2-2.5 units/g of CDW (Table III). It can be concluded that at least
95% of acid formed by induced E. coli JM101 (pSPZ3) is due
to the presence of XMO. No significant differences with respect to
products formed and rates of formation between the routinely used
inducer, 0.1% (v/v) n-octane, and the alternatively used
0.05% (v/v) DCPK were observed (results not shown).
18O Incorporation Experiments--
These experiments
were done to determine how in the above reactions alcohols, aldehydes,
and acids are formed from toluene, pseudocumene, and possibly also
other substituted toluenes. One possibility is the consecutive
incorporation of single atoms from dioxygen into a methyl substituent
of the aromatic ring via a monooxygenase reaction. A second possibility
is that aldehydes and acids are formed from benzyl alcohol and
3,4-dimethylbenzyl alcohol via oxidation reactions catalyzed by dehydrogenases.
Toluene, pseudocumene, and the corresponding alcohols and aldehydes
were incubated with whole cells of E. coli JM101 (pSPZ3) with normal air or an 18O2-enriched atmosphere
(85 atom % 18O) as described under "Materials and
Methods." Following incubation, reaction mixtures were extracted with
diethyl ether and analyzed by gas chromatography-mass spectrometry.
Products showing mass spectra characteristic of benzyl alcohol,
benzaldehyde, benzoic acid, 3,4-dimethylbenzyl alcohol,
3,4-dimethylbenzaldehyde, and 3,4-dimethylbenzoic acid have been
detected. Fig. 4 (A and
B) shows the mass spectra of 3,4-dimethylbenzyl alcohol
formed from pseudocumene after incubation with air (A) and
with an 18O2-enriched atmosphere
(B). The fragmentation patterns of the two spectra clearly
show that one atom of dioxygen was incorporated into pseudocumene
during the course of the reaction. The ratio of the M+ +2
to M+ molecular ion peaks (Fig. 4B) is 85:15 for
both the m/z 136 (3,4-dimethylbenzyl alcohol) and
the m/z 121 (methyl-benzyl alcohol species)
peaks.
We observed a rapid exchange of oxygen during incubation of
benzaldehyde and 3,4-dimethylbenzaldehyde with
H218O (60 atom % 18O), the
exchange being faster for benzaldehyde than for
3,4-dimethylbenzaldehyde. After 5 and 20 min of incubation, 40 and
50%, respectively, of the oxygen in benzaldehyde was 18O.
For 3,4-dimethylbenzaldehyde these values amounted to 11 and 25%. This
rapid oxygen exchange in aldehydes precluded a quantitative measurement
of 18O incorporation into the aldehydes during the
oxidation of toluene, pseudocumene, and the corresponding alcohols by
XMO.
Nevertheless, the mass spectra of 3,4-dimethylbenzaldehyde formed from
3,4-dimethylbenzyl alcohol (Fig. 4, C and D)
showed a minor enrichment of 18O in the aldehyde (2.1%)
for the reaction under an 18O2-enriched
atmosphere (D). Assuming a monooxygenase reaction for the
oxidation of the alcohols with the formation of a diol followed by
spontaneous unspecific dehydration 42.5% M+ +2 ion
enrichment would be expected in the absence of oxygen exchange with
solvent water. When pseudocumene was added as substrate, after two
oxygenation steps only 5% enrichment of the M+ +2 ion was
observed for 3,4-dimethylbenzaldehyde (expected: 72%) (results not
shown). This low level of 18O enrichment in the aldehyde
can again be explained by the rapid exchange of oxygen in aldehydes.
When 3,4-dimethylbenzaldehyde was added as substrate in an
18O2-enriched atmosphere, the molecular ion
value of the emerging acid increased by 2 atomic mass units over the
value obtained in air (results not shown). In this case the ratio
between M+ +2 and M+ was 85:15, indicating that
the XMO-catalyzed formation of the acid from the aldehyde requires the
introduction of one oxygen atom from molecular oxygen. When two such
monooxygenation steps are involved in the XMO-catalyzed formation of
3,4-dimethylbenzoic acid from 3,4-dimethylbenzyl alcohol, the
incorporation of two 18O atoms into the acid is expected in
an 18O2-enriched atmosphere. Ignoring the rapid
exchange of oxygen in aldehydes, this would result in a ratio of
8.6:55.3:36.1 between the three molecular ion values 150, 152, and 154 of the formed acid. We indeed observed the incorporation of two
18O atoms into the acid in an
18O2-enriched atmosphere (Fig. 4, E
and F). The ratio of the three normalized molecular ion
values 150, 152, and 154 was 10:80:10 (F).
For toluene and its derivatives as substrates and products, analogous
results were obtained. Again, formation of benzyl alcohol from toluene
and of benzoic acid from benzaldehyde in an
18O2-enriched atmosphere resulted in an
increase of the molecular ion value by 2 atomic mass units. Because of
the already mentioned fact that the oxygen exchange in benzaldehyde is
faster than in 3,4-dimethylbenzaldehyde, the intensity of the observed
M+ +2 ion of benzaldehyde formed from toluene under
18O2-enriched atmosphere was lower (3.4%) than
for 3,4-dimethylbenzaldehyde formed from pseudocumene. For benzaldehyde
formed from benzyl alcohol the M+ +2 ion was completely
absent (results not shown).
Kinetics of Oxygenation Reactions in E. coli JM101(pSPZ3)--
To follow product formation over time, cells
were incubated with the same substrate for different time periods. When
toluene or pseudocumene were added as substrates, the consecutive
accumulation of the corresponding alcohols, aldehydes, and acids was
observed (Figs. 5A and
6A). As also found in the
activity assays, benzyl alcohol, 3,4-dimethylbenzyl alcohol, and
benzaldehyde were formed at high rates, 3,4-dimethylbenzaldehyde and
3,4-dimethylbenzoic acid were formed at intermediate rates, and benzoic
acid accumulated slowly. In the first 5 min products were formed from
either pseudocumene or toluene with a specific activity of 100 units/g of CDW (Table II). Between 5 and 10 min benzaldehyde was formed
at a rate of 80 units/g of CDW, whereas 3,4-dimethylbenzaldehyde was
formed more slowly (37 units/g of CDW). Acid formation started when
toluene or pseudocumene had completely disappeared, at rates of 3.2 and 21 units/g of CDW for benzoic acid and 3,4-dimethylbenzoic acid, respectively, between 10 and 30 min. A low level of benzyl alcohol always remained. Between 40 and 80 min, the level of benzyl alcohol started to increase again, whereas the benzaldehyde level decreased (Fig. 5A). Pseudocumene, 3,4-dimethylbenzyl alcohol and
3,4-dimethylbenzaldehyde were completely used up to form
3,4-dimethylbenzoic acid (Fig. 6A).
When 3,4-dimethylbenzyl alcohol was added as the substrate,
3,4-dimethylbenzaldehyde was formed at a rate of 50 units/g of CDW
(Table II), whereas 3,4-dimethylbenzoic acid was formed at a rate of 23 units/g of CDW between 10 and 30 min (Fig. 6C). With benzyl
alcohol as a substrate the benzaldehyde formation rate amounted to 95 units/g of CDW, and benzoic acid formed at a constant rate of 2.9 units/g of CDW (results not shown). In contrast to benzyl alcohol,
3,4-dimethylbenzyl alcohol was completely converted to the acid in this
same time period.
3,4-Dimethylbenzoic acid was formed at a rate of 55 units/g of CDW
(Table II) when 3,4-dimethylbenzaldehyde was added as substrate (Fig.
6E), and after 20 min it already was the predominant
species. Slow and constant formation of benzoic acid (3 units/g of CDW) was observed when benzaldehyde was added as substrate (results not
shown). Here, the initial rate in the first 5 min was 10 units/g of CDW
(Table II).
Conversions of Toluene and Pseudocumene by E. coli JM101(pRMAB)--
We tested whether the presence of BADH
together with XMO increased or decreased the rate of aldehyde formation
from toluene and pseudocumene. The latter is a distinct possibility
because several other alcohol dehydrogenases catalyze reactions with an equilibrium on the side of the alcohol at physiological pH.
Consequently BADH might decrease the net rate of aldehyde formation. On
the other hand a two-step oxidation of toluene (pseudocumene) via benzyl alcohol (3,4-dimethylbenzyl alcohol) to benzaldehyde
(3,4-dimethylbenzaldehyde) catalyzed by XMO in concert with BADH might
result in increased formation rates of the aldehydes since reduced
nicotinamide cofactors oxidized by XMO would be regenerated by BADH. To
clarify these questions we cloned the BADH gene xylB into
pSPZ3 directly downstream of the xylA gene. The resulting
plasmid was called pRMAB (Fig. 2).
Biotransformations by E. coli JM101 carrying this plasmid
were in fact clearly different from those by E. coli JM101
(pSPZ3). Toluene was transformed to its corresponding products with an initial specific activity of 87 units/g of CDW. The corresponding values for benzyl alcohol, pseudocumene, and 3,4-dimethylbenzyl alcohol
were 66, 73, and 42 units/g of CDW, respectively. The presence of BADH
clearly reduced the level of oxidation products formed by XMO. Here
also we followed product formation over 80 min. When toluene was added,
we observed consecutive accumulation of benzyl alcohol, benzaldehyde,
and benzoic acid (Fig. 5B). Between 5 and 10 min
benzaldehyde was formed at a rate of 34 units/g of CDW, and the acid
accumulated at a rate of 1 unit/g of CDW between 10 and 40 min. After
about 20 min the level of benzyl alcohol started to increase again.
After 80 min almost no benzaldehyde remained, and a very low amount of
benzoic acid was formed. When benzyl alcohol was added as a substrate,
analogous results were obtained (results not shown); the level of the
aldehyde decreased after about 20 min, whereas the alcohol was formed.
Clearly, expression of BADH together with XMO caused a considerable
reduction of benzaldehyde to benzyl alcohol, indicating that formation
of benzaldehyde is not enhanced by additional BADH.
When pseudocumene was added as a substrate, we again observed
consecutive accumulation of 3,4-dimethylbenzyl alcohol,
3,4-dimethylbenzaldehyde, and 3,4-dimethylbenzoic acid (Fig.
6B). Between 5 and 10 min 3,4-dimethylbenzaldehyde was
formed at a rate of 15 units/g of CDW. The acid accumulated at about
the same rate (13 units/g of CDW) between 30 and 40 min. After the
rapid formation of the alcohol, the aldehyde level remained quite high
for 20 min. Nevertheless, at the end of the reaction, alcohol and
aldehyde were completely transformed to the acid. 3,4-Dimethylbenzaldehyde and 3,4-dimethylbenzoic acid were formed when
3,4-dimethylbenzyl alcohol was added as substrate (Fig. 6D). Between 20 and 30 min, the acid was formed with a specific activity of
8 units/g of CDW, and the aldehyde concentration never exceeded the
alcohol concentration. The continued presence of 3,4-dimethylbenzyl alcohol indicates that besides the oxygenations there is a simultaneous reduction of aldehyde to alcohol for which BADH seems to be responsible.
The alk Regulatory System as an Expression System for
XMO--
E. coli recombinants expressing XylMA have been
used to oxidize styrene to (S)-styrene oxide on a 2L reactor
scale (27, 28). However, activity has been limited, and this is
probably due to insufficient expression of the xyl genes in
E. coli. The alk regulatory system of P. oleovorans GPo1 is not subject to catabolite repression in
E. coli, and expression of the alk genes in
glucose grown E. coli W3110 via the alk
regulatory system permits accumulation of the membrane located alkane
hydroxylase AlkB up to 10-15% of total cell protein (29, 30). The
alk regulatory system has therefore been used to increase
the volumetric activities of E. coli recombinants producing
(S)-styrene, based on a 5-fold increase of the expression of
xylMA via the alk regulatory system, resulting in
styrene oxidation activities of up to 91 units/g of CDW (21).
In the present study we used related constructs and reached specific
activities between 100 and 120 units/g of CDW for toluene and
pseudocumene as substrates. Compared with earlier studies with E. coli expressing XylMA this corresponds to a 10-20-fold activity
increase (20, 22). Cells were rapidly induced by both
n-octane and DCPK, but n-octane was a better
inducer than DCPK, because with n-octane maximal induction
is reached at lower concentrations than with DCPK (Fig. 3). Induced
cells grew more slowly, indicating a stress effect either of the gene
products or of the inducers themselves. Growth was reduced most when
XMO expression was maximal. This stress effect has been described before (20, 31). Additionally, DCPK concentrations in excess inhibit
cell growth. For n-octane in water this is not the case, most probably because of its very low solubility.
XMO Catalyzes the Oxygenation Not Only of Toluene and Xylenes but
Also of the Corresponding Alcohols and Aldehydes--
The results
obtained in the present study show that XMO has aromatic alcohol and
aldehyde oxidation activities to form the corresponding acids via
monooxygenation reactions. Uninduced E. coli JM101 (pSPZ3)
and induced E. coli JM101 (pRS) as controls did not carry
out these biotransformations.
The oxidation of benzyl alcohols by XMO has been reported by Harayama
et al. (2, 22), but in a later study including in
vitro experiments the authors concluded that XMO is not
responsible for such activity (9). Instead chromosomally encoded
dehydrogenases of the E. coli host, which transform benzyl
alcohol to benzoate, were supposed to be responsible for the
transformation of benzyl alcohol to benzoate (2). But the incorporation
of 18O into the products, observed in this study and
discussed below, provides strong evidence for a monooxygenation type of
reaction catalyzed by XMO.
In control experiments, in which aldehydes were incubated with
uninduced E. coli JM101 (pSPZ3) and induced E. coli JM101 (pRS), we observed the reduction of aldehydes to
alcohols. These transformations, rather than the oxidation of alcohols,
can be explained by the action of the E. coli alcohol
dehydrogenases that catalyze an equilibrium lying, for thermodynamic
reasons, on the side of the alcohols. The net formation of benzyl
alcohol in the end of the biotransformation of toluene by E. coli JM101 (pSPZ3) (Fig. 5A) can also be attributed to
E. coli dehydrogenases, and this activity becomes
significant because XMO loses activity with time.
Two significant differences were observed between the consecutive
oxygenation of toluene and pseudocumene. 3,4-Dimethylbenzyl alcohol
is oxygenated more slowly than benzyl alcohol, and 3,4-dimethylbenzoic acid is formed at a clearly higher rate than benzoic acid. This indicates that there are differences in the effect of varying substitutions on the specific activities of XMO toward oxidized substrates (alcohols and aldehydes) compared with that toward unoxidized substrates like toluene and pseudocumene, which are hydroxylated at a very similar rate.
Another interesting finding is that the acids were not formed until
toluene or pseudocumene had disappeared more or less completely. This
points to a higher affinity of XMO for toluene and pseudocumene than
for the corresponding aldehydes.
The Presence of BADH Results in the Back Formation of Benzyl
Alcohol from Benzaldehyde--
In cells containing BADH the aldehydes
accumulate at a clearly lower rate. BADH seems to drastically increase
the effect of the E. coli dehydrogenases; the equilibrium of
this dehydrogenase reaction seems to lie on the side of the alcohol. In
fact, the Gibbs energy change of the dehydrogenation of benzyl alcohol, calculated according to the group contribution method of Mavrovouniotis (32, 33), is 5 kcal/mol or, calculated according to the Gibbs energies
of formation given by Dean (34), 4 kcal/mol. (The Gibbs energy change
of the oxygenation of benzyl alcohol amounts to
After only 20 min there was net formation of benzyl alcohol by E. coli JM101 (pRMAB). This was not observed for E. coli
JM101 (pSPZ3) as the biocatalyst (Fig. 5B).
Here, the following question arises: what could be the physiological
roles of BADH and BZDH in the natural host P. putida (pWW0),
when XMO alone is able to catalyze all reactions from the unoxidized
substrates to the carboxylic acids? Concerning BZDH the answer seems to
be evident: in the absence of BZDH and in the presence of unoxidized
substrate (e.g. toluene and pseudocumene), the corresponding
aldehydes would not be oxidized because of their low affinity to XMO.
In addition, for benzaldehyde the oxygenation rate is quite low, which
could also be the case for other aromatic aldehydes. Thus, BZDH may be
necessary for the efficient formation of acids in its native host. When
the fluxes from the alcohols to the acids and through the
meta cleavage pathway are high, which again requires highly
active BZDH, the concentrations of intermediates like benzaldehyde
would be very low, and BADH could also catalyze a part or even most of
the aldehyde formation. The dehydrogenation of alcohols and aldehydes
is favorable for the host because reducing equivalents are produced
rather than consumed, as is the case for the first oxygenation step. In
addition, accumulation of these toxic intermediates in the cytosol is
prevented. In the case of high concentrations of these intermediates
BADH could, by aldehyde reduction, preserve low concentrations of the
particularly reactive aldehydes.
The Most Probable Mechanism for Alcohol Oxidation Includes the
Formation of a gem-Diol as an Intermediate--
The quantitative
18O incorporation into aromatic alcohols and acids
characterized the formation of these compounds as monooxygenation reactions. Alcohol oxidation to carbonyl products is usually mediated by pyridine nucleotide-dependent dehydrogenases like BADH.
However, XMO catalyzes the NADH-dioxygen-dependent
oxidation of benzyl alcohol and 3,4-dimethylbenzyl alcohol to the
corresponding aldehydes (Table II). Similar reactions are also
catalyzed by the alkane hydroxylase of P. oleovorans, which
was reported to catalyze the oxygenative formation of medium chain
length alkanals from terminal alkanols (36), and by ammonia-grown cells
of Nitrosomonas europea, which also oxidizes toluene through
benzyl alcohol to benzaldehyde (37). Rabbit liver cytochrome P-450
(forms 2B4 and 2E1) (38) and purified naphthalene dioxygenase from
Pseudomonas sp. strain NCIB 9816-4 (39) were reported to
oxidize benzyl alcohol and 1-phenylethyl alcohol. Because of the rapid
exchange of oxygen in aldehydes, both studies concentrated on the
1-phenylethyl alcohol oxidation to evaluate possible mechanisms. Vaz
and Coon (38) suggested that the mentioned forms of cytochrome P-450
oxidize the alcohol to a carbon radical followed by coupling of oxygen to form a gem-diol intermediate that undergoes
nonstereospecific dehydration to yield acetophenone. Another possible
mechanism, not utilized by either of the two cytochrome P-450 forms,
involves desaturation to form an enol intermediate followed by
tautomerization to acetophenone. In the case of naphthalene dioxygenase
and 1-phenylethyl alcohol, despite the fact that no 18O
from molecular dioxygen was found to be incorporated into acetophenone, Lee and Gibson (39) suggested that both mechanisms could contribute to
product formation. They argued that stereospecific dehydration of
putative gem-diol intermediates, which has been proposed for other oxidations (40, 41), could occur with the aid of a basic amino
acid residue in the active site of the enzyme (38). Lee and Gibson as
well as Vaz and Coon suggested that the formation of a
gem-diol intermediate could account for the oxidation of benzyl alcohol to benzaldehyde by naphthalene dioxygenase and the two
forms of cytochrome P-450, respectively. In the case of XMO the signals
for the M+ +2 ion in the mass spectrum of
3,4-dimethylbenzaldehyde (2.1%) (Fig. 4D) and for the
M+ +4 ion in the mass spectrum of 3,4-dimethylbenzoic acid
(10%) (Fig. 4F), formed from the corresponding alcohol,
indicate the monooxygenation of the alcohol and as a result the
formation of a gem-diol intermediate followed by
nonstereospecific dehydration yielding the aldehyde (Fig.
7). After incubation of
3,4-dimethylbenzaldehyde in water containing 60%
H218O under conditions similar to those of the
enzymatic reaction, but with 3,4-dimethylbenzyl alcohol, potassium
phosphate, glucose, and the cells omitted, only 25% of the oxygen was
exchanged. Therefore it can not be completely excluded that
stereospecific dehydration also contributes to aldehyde formation. The
results presented in this study strongly suggest that the most probable
mechanism for alcohol oxidation by XMO includes the formation of a
gem-diol as an intermediate.
The ability of XMO to catalyze three consecutive oxygenation steps at
high rates makes it a very interesting non-heme iron enzyme. The
in vitro characterization of this enzyme is an approach that, based on the high activity of XMO, will hopefully allow a
detailed analysis of this interesting reaction.
We thank Sven Panke for providing plasmids
and for helpful discussions and Hans Peter E. Kohler for the gift of
18O2.
*
This work was supported by the BASF corporation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Inst. für
Biotechnologie, ETH Zürich, Hönggerberg HPT, CH-8093
Zürich, Switzerland. Tel.: 41-1-633-36-91; Fax: 41-1-633-10-51;
E-mail: andreas@biotech.biol.ethz.ch.
The abbreviations used are:
XMO, xylene
monooxygenase;
BADH, benzyl alcohol dehydrogenase;
BZDH, benzaldehyde
dehydrogenase;
DCPK, dicyclopropylketone;
CDW, cell dry weight;
HPLC, high performance liquid chromatography;
GC, gas chromatography;
MS, mass spectrometry.
Xylene Monooxygenase Catalyzes the Multistep Oxygenation of
Toluene and Pseudocumene to Corresponding Alcohols, Aldehydes, and
Acids in Escherichia coli JM101*
,§,
Institute of Biotechnology, Swiss Federal
Institute of Technology Zurich, CH-8093 Zurich, Switzerland and the
¶ BASF Corporation, Research Fine Chemicals and Biotechnology,
D-67056 Ludwigshafen, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Upper TOL pathway. The consecutive
oxidation of toluene to benzyl alcohol, benzaldehyde, and benzoic acid
by enzymes of the upper TOL pathway and the organization of the
xyl genes of the upper TOL operon are shown.
Pu, upper TOL operon promoter; xylW,
gene with unknown function; xylC, gene encoding BZDH;
xylM, gene encoding the terminal hydroxylase component of
XMO; xylA, gene encoding the NADH:acceptor reductase
component of XMO; xylB, gene encoding BADH; xylN,
gene with unknown function.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids
-D-1-thiogalactopyranoside. Solid media
contained 1.5% (w/v) agar. Liquid cultures were routinely incubated on
horizontal shakers at 200 rpm and 30 or 37 °C.
View larger version (11K):
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Fig. 2.
Expression plasmids pSPZ3 and pRMAB;
xylMA and xylMAB under the control of
the alk regulatory system. alkBp,
promoter of the alk operon; alkS, gene for the
positive regulator AlkS. xylM* and xylA, genes coding for
the xylene monooxygenase (* indicates that in xylM a
NdeI site is removed); xylB, gene encoding BADH;
Km, kanamycin resistance gene; T4t, phage
T4 transcriptional terminator.
5-1% (v/v) for induction tests), and the incubation
was continued for 3-3.5 h, at which time A450
had typically increased to 0.8-0.9. The cells were harvested and
resuspended to a cell dry weight (CDW) of 2.5 g/liter in 50 mM potassium phosphate buffer, pH 7.4, containing 1% (w/v)
glucose. Aliquots of 1 or 2 ml were distributed in stoppered Pyrex
tubes and incubated horizontally on a rotary shaker at 250 rpm and
30 °C. After 5 min, substrate was added to a final concentration of
1.5 mM from a 20-fold stock solution in ethanol. For
experiments in which the formation of 3,4-dimethylbenzoic acid was
expected, CDW was reduced to 1 g/liter, and substrates were added to a
final concentration of 0.5 mM. The reaction was carried out
for 5 min in the shaker and then stopped by placing the samples in ice
and immediately adding 40 or 80 µl of a perchloric acid stock
solution (10% v/v) to bring the suspension to pH 2.
1 CDW). Experiments were repeated at least three
times independently.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 3.
Growth and induction kinetics of XMO in
E. coli JM101 (pSPZ3). A shows the XMO
activity and the CDW at different time intervals after induction by
n-octane, whereas B and C show the
same values 3.5 h after induction by different amounts of
n-octane and DCPK, respectively. For each activity point a
single culture was grown. The activity assays were performed as
described under "Materials and Methods." Pseudocumene (1.37 mM) was added to a suspension of resting cells of E. coli JM101 (pSPZ3) (2.04-2.44 g liter 1 CDW) in 50 mM potassium phosphate buffer, pH 7.4, containing 1% (w/v)
glucose. The specific activities are based on product formation during
the initial 5 min of reaction. The arrow in A
indicates when 0.1% (v/v) n-octane was added to induce
XylMA synthesis. 
, CDW of uninduced cultures; 
, CDW of induced
cultures; ×, specific activities of induced cultures.
Oxidation of toluene and derivatives by xylene monooxygenase
Transformations of aldehydes in control experiments
View larger version (17K):
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Fig. 4.
Mass spectra of products formed by E. coli JM101 (pSPZ3) in air (A,
C, and E) and in an
18O2-enriched atmosphere (B,
D, and F). A and
B show the mass spectrometry analysis of 3,4-dimethylbenzyl
alcohol produced from pseudocumene, C and D show
the analysis of 3,4-dimethylbenzaldehyde also produced from
3,4-dimethylbenzyl alcohol, and E and F show the
analysis of 3,4-dimethylbenzoic acid produced from 3,4-dimethylbenzyl
alcohol.
View larger version (16K):
[in a new window]
Fig. 5.
Oxidation of toluene by E. coli
JM101 (pSPZ3) (A) and E. coli
JM101 (pRMAB) (B). The assay was performed
as described under "Materials and Methods." Toluene (1.37 mM) was added to a suspension of resting E. coli
JM101 (pSPZ3/pBRMAB) (2.07-2.14 g liter 1 CDW) in 50 mM potassium phosphate buffer, pH 7.4, containing 1% (w/v)
glucose. , toluene; 
, benzyl alcohol; 
, benzaldehyde; 
,
benzoic acid; ×, sum of the four concentrations.
View larger version (29K):
[in a new window]
Fig. 6.
Oxidation of pseudocumene, the corresponding
alcohol, and the corresponding aldehyde by E. coli
JM101 (pSPZ3) (A, C, and
E) and E. coli JM101 (pRMAB)
(B and D). The assay was
performed as described under "Materials and Methods." Substrates
(0.46 mM) were added to a suspension of resting E. coli JM101 (pSPZ3/pBRMAB) (0.86-0.92 g liter 1 CDW)
in 50 mM potassium phosphate buffer, pH 7.4, containing 1%
(w/v) glucose. A and B show the oxidation of
pseudocumene, C and D show the oxidation of
3,4-dimethylbenzyl alcohol, and E shows the oxidation of
3,4-dimethylbenzaldehyde. , pseudocumene; 
, 3,4-dimethylbenzyl
alcohol; 
, 3,4-dimethylbenzaldehyde; 
, 3,4-dimethylbenzoic acid;
×, sum of the four concentrations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 kcal/mol.) On the
basis of these values an equilibrium constant between benzyl alcohol
and benzaldehyde of around 103 can be assumed. The
estimated ratio between the equilibrium concentrations of benzaldehyde
and benzyl alcohol including an NAD/NADH ratio of 10.6 in E. coli under aerobic conditions and glucose excess (35) amounts to
1:100. In addition, BADH was reported to have lower
Km and higher Vmax values for
the reverse reaction (from aldehydes to alcohols) compared with the
forward reaction, whereas the optimal pH, at which the measurements
were performed, was 9.4 for the forward and 5.7 for the reverse
reaction (16-18). For benzyl alcohol and benzaldehyde Shaw et
al. (18) found Km values of 155 and 65 µM and Vmax values of 320 and 4800 µmol min1 mg1 for the forward and the
reverse reactions, respectively, in 100 mM glycine.
View larger version (10K):
[in a new window]
Fig. 7.
Monooxygenation of benzyl alcohol by XMO;
proposed formation of a gem-diol. XMO
indicates xylene monooxygenase of P. putida mt-2 expressed
in E. coli JM101 (pSPZ3).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Harayama, S.,
Kok, M.,
and Neidle, E. L.
(1992)
Annu. Rev. Microbiol.
46,
565-601[CrossRef][Medline]
[Order article via Infotrieve]
2.
Harayama, S.,
Rekik, M.,
Wubbolts, M.,
Rose, K.,
Leppik, R. A.,
and Timmis, K. N.
(1989)
J. Bacteriol.
171,
5048-5055 3.
Abril, M.-A.,
Michan, C.,
Timmis, K. N.,
and Ramos, J. L.
(1989)
J. Bacteriol.
171,
6782-6790 4.
Williams, P. A.,
Shaw, L. M.,
Pitt, C. W.,
and Vrecl, M.
(1997)
Microbiology
143,
101-107 5.
Ramos, J. L.,
Marqués, S.,
and Timmis, K. N.
(1997)
Annu. Rev. Microbiol.
51,
341-373[CrossRef][Medline]
[Order article via Infotrieve]
6.
Suzuki, M.,
Hayakawa, T.,
Shaw, J. P.,
Rekik, M.,
and Harayama, S.
(1991)
J. Bacteriol.
173,
1690-1695 7.
Shaw, J. P.,
and Harayama, S.
(1992)
Eur. J. Biochem.
209,
51-61[Medline]
[Order article via Infotrieve]
8.
Wubbolts, M.
(1994)
Xylene and Akane Mono-oxygenases from Pseudomonas putida.Ph.D. thesis
, Rijksuniversiteit, Groningen
9.
Shaw, J. P.,
and Harayama, S.
(1995)
J. Ferm. Bioeng.
79,
195-199[CrossRef]
10.
Baptist, J. N.,
Gholson, R. K.,
and Coon, M. J.
(1963)
Biochem. Biophys. Acta
69,
40-47
11.
Chakrabarty, A. M.,
Chou, G.,
and Gunsalus, L. G.
(1973)
Proc. Natl. Acad. Sci. U. S. A.
70,
1137-1140 12.
Kok, M.,
Oldenhuis, R.,
van der Linden, M. P. G.,
Raatjes, P.,
Kingma, J.,
van Lelyveld, P. H.,
and Witholt, B.
(1989)
J. Biol. Chem.
264,
5435-5441 13.
van Beilen, J. B.,
Wubbolts, M. G.,
and Witholt, B.
(1994)
Biodegradation
5,
161-174[CrossRef][Medline]
[Order article via Infotrieve]
14.
Shanklin, J.,
Whittle, E.,
and Fox, B. G.
(1994)
Biochemistry
33,
12787-12794[CrossRef][Medline]
[Order article via Infotrieve]
15.
Shanklin, J.,
Achim, C.,
Schmidt, H.,
Fox, B. G.,
and Münck, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2981-2986 16.
Shaw, J. P.,
and Harayama, S.
(1990)
Eur. J. Biochem.
191,
705-714[Medline]
[Order article via Infotrieve]
17.
Shaw, J. P.,
Schwager, F.,
and Harayama, S.
(1992)
Biochem. J.
283,
789-794
18.
Shaw, J. P.,
Rekik, M.,
Schwager, F.,
and Harayama, S.
(1993)
J. Biol. Chem.
268,
10842-10850 19.
Kunz, D. A.,
and Chapman, P. J.
(1981)
J. Bacteriol.
146,
179-191 20.
Wubbolts, M. G.,
Reuvekamp, P.,
and Witholt, B.
(1994)
Enzyme Microb. Technol.
16,
608-615[CrossRef][Medline]
[Order article via Infotrieve]
21.
Panke, S.,
Meyer, A.,
Huber, C. M.,
Witholt, B.,
and Wubbolts, M. G.
(1999)
Appl. Environ. Microbiol.
65,
2324-2332 22.
Harayama, S.,
Leppik, R. A.,
Rekik, M.,
Mermod, N.,
Lehrbach, P. R.,
Reineke, W.,
and Timmis, K. N.
(1986)
J. Bacteriol.
167,
455-461 23.
Grund, A.,
Shapiro, J.,
Fennewald, M.,
Bacha, P.,
Leahy, J.,
Markbreiter, K.,
Nieder, M.,
and Toepfer, M.
(1975)
J. Bacteriol.
123,
546-556 24.
Yuste, L.,
Canosa, I.,
and Rojo, F.
(1998)
J. Bacteriol.
180,
5218-5226 25.
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
26.
Panke, S.,
Witholt, B.,
Schmid, A.,
and Wubbolts, M. G.
(1998)
Appl. Environ. Microbiol.
64,
2032-2043 27.
Wubbolts, M. G.,
Hoven, J.,
Melgert, B.,
and Witholt, B.
(1994)
Enzyme Microb. Technol.
16,
887-893[CrossRef]
28.
Wubbolts, M. G.,
Favre-Bulle, O.,
and Witholt, B.
(1996)
Biotechnol. Bioeng.
52,
301-308[CrossRef]
29.
Nieboer, M.,
Kingma, J.,
and Witholt, B.
(1993)
Mol. Microbiol.
8,
1039-1051[CrossRef][Medline]
[Order article via Infotrieve]
30.
Staijen, I. E.,
Marcionelli, R.,
and Witholt, B.
(1999)
J. Bacteriol.
181,
1610-1616 31.
Nieboer, M.
(1996)
Over production of a Foreign Membrane Monooxygenase in E. coli: Relocation to Membrane Biogenesis.Ph.D. thesis
, Rijksuniversiteit, Groningen
32.
Mavrovouniotis, M. L.
(1990)
Biotechnol. Bioeng.
36,
1070-1082[CrossRef]
33.
Mavrovouniotis, M. L.
(1991)
J. Biol. Chem.
266,
14440-14445 34.
Dean, J. A.
(ed)
(1985)
Lange's Handbook of Chemistry
, 13th Ed.
, McGraw-Hill Book Co., New York
35.
Leonardo, M. R.,
Dailly, Y.,
and Clark, D. P.
(1996)
J. Bacteriol.
178,
6013-6018 36.
May, S. W.,
and Katopodis, A. G.
(1986)
Enzyme Microb. Technol.
8,
17-21
37.
Keener, W. K.,
and Arp, D. J.
(1994)
Appl. Environ. Microbiol.
60,
1914-1920 38.
Vaz, A. D. N.,
and Coon, M. J.
(1994)
Biochemistry
33,
6442-6449[CrossRef][Medline]
[Order article via Infotrieve]
39.
Lee, K.,
and Gibson, D. T.
(1996)
Appl. Environ. Microbiol.
62,
3101-3106[Abstract]
40.
Spain, J. C.,
Zylstra, G. J.,
Blake, C. K.,
and Gibson, D. T.
(1989)
Appl. Environ. Microbiol.
55,
2648-2652 41.
Robertson, J. B.,
Spain, J. C.,
Haddock, J. D.,
and Gibson, D. T.
(1992)
Appl. Environ. Microbiol.
58,
2643-2648 42.
Hanahan, D.
(1983)
J. Mol. Biol.
166,
557-580[Medline]
[Order article via Infotrieve]
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