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Previous Article | Next Article 
Appl Environ Microbiol, February 1998, p. 748-751, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Engineering of Quasi-Natural Pseudomonas
putida Strains for Toluene Metabolism through an
ortho-Cleavage Degradation Pathway
Sven
Panke,
Juan M.
Sánchez-Romero, and
Víctor
de Lorenzo*
Centro Nacional de
Biotecnología
CSIC, Campus de Cantoblanco, Madrid 28049, Spain
Received 12 May 1997/Accepted 21 September 1997
 |
ABSTRACT |
To construct a bacterial catalyst for bioconversion of toluene and
several alkyl and chloro- and nitro-substituted derivatives into the
corresponding benzoates, the upper TOL operon of plasmid pWW0 of
Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The
corresponding DNA segment was then targeted to the chromosome of a
P. putida strain by using a genetic technique that allows
deletion of all recombinant tags inherited from previous cloning steps
and leaves the otherwise natural strain bearing exclusively the DNA
segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes,
and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.
| |
TEXT |
Many Pseudomonas strains
are able to employ as their sole carbon source a variety of unusual
chemicals, including a wide range or aromatic hydrocarbons and their
derivatives (25, 30). The catabolic pathways involved (often
encoded by plasmids) are composed of distinct steps, each of which can
be split from the others with the tools of recombinant DNA and
expressed independently (28). Not infrequently, the enzymes
involved in one or more steps in a catabolic pathway have activity on
structural analogs of the natural substrates and can thereby give rise
to derivatives of interest for industry (29). This is the
case for certain enzymes included in the catabolic pathway for toluene,
m-xylene, p-xylene, and ethyl benzene, encoded by
the TOL plasmid pWW0 of Pseudomonas putida mt-2 (14,
21). Degradation of toluene through the TOL pathway has two major
steps, namely, bioconversion of this substrate into benzoate and
subsequent catabolism of its aromatic ring down to Krebs cycle
metabolites via a catechol intermediate that is cleaved in
meta by one of the pathway enzymes. The enzymes that
determine conversion of toluene to benzoate are encoded by the
so-called upper pathway (Fig. 1), and
they happen to have activity not only on the hydrocarbon but also on a
variety of chloro and nitro derivatives (1-3). Therefore,
the expression of the collective enzymes of the upper pathway away from
the remaining TOL genes is predicted to yield a collection of nitro-
and halo-substituted benzoates. However, the development of a bacterial
catalyst for these biotransformations has been impossible because of
the difficulty in reassembling the entire upper TOL operon along with
its cognate regulatory gene (xylR; 21),
away from the meta-cleavage activity.
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FIG. 1.
Biodegradation of toluene by P. putida
through alternative meta or ortho lower pathways.
P. putida strains carrying the TOL plasmid pWW0 perform
sequential oxidation of the CH3 group of toluene to
alcohol, aldehyde, and acid through the action of the enzymes xylene
monooxygenase (encoded by xylAM), benzyl alcohol
dehydrogenase (encoded by xylB), and benzaldehyde
dehydrogenase (encoded by xylC), respectively (14,
21). The resulting benzoate is then channeled into a lower route
involving meta-ring cleavage of the catechol intermediate
through the action of the products of the TOL genes xylXYZL,
xylE, and xylFKJQ, all the way down to Krebs
cycle intermediates (TCA, tricarboxylic acid cycle). In the absence of
the TOL meta pathway, benzoate can be channeled through an
alternative, chromosomally encoded route (involving the ben
genes) for formation of catechol (30). This is then
subjected to ortho-ring cleavage by the product of the
catA gene (catechol 1,2-dioxygenase) and further converted
to metabolic intermediates. The meta-cleavage pathway is
generally incompatible with the degradation of chloroaromatics (2,
30).
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A more general problem of using recombinant strains in
biotransformations or bioremediation of aromatic hydrocarbons is the instability of the cloned genes (frequently borne by plasmids) and the
inheritance of marker genes (antibiotic or not antibiotic) used for
selection (4, 5). The presence of antibiotic resistance genes in strains destined for environmental release is particularly undesirable under U.S. and European Union regulations (5). While the problem of stability has been alleviated by the use since
1990 of Tn5-derived transposon vectors for stable
chromosomal insertion of cloned genes (6, 7, 16), the
resulting strains still inherit DNA segments unrelated to the desired
phenotype (i.e., those of the selection markers included in the
transposon vector). As a consequence, the resulting biocatalysts are
still quite different from those which could arise during natural
evolution, by processes involving exclusively shuffling of DNA segments
through transposition (27, 30) or DNA slippage
(15).
In this work, we have employed transposon vectors with excisable
selection markers (18) for insertion of the reconstructed and fully functional upper TOL pathway into the chromosome of P. putida KT2442 (7). The resulting strains convert
toluene into benzoate and further metabolize this compound through the housekeeping ortho-ring cleavage pathway of the catechol
intermediate. Due to the loss of recombinant markers during the
construction process, these genetically engineered strains differ
minimally from their nonrecombinant counterparts.
Reconstruction of the upper TOL operon as a single catabolic
segment.
A gene cassette bearing the complete upper operon along
with its native toluene-responsive regulator gene, xylR, was
constructed by using the strategy shown in Fig.
2. The origin of the upper TOL sequences
was plasmids pED3306 and pRL4, which span overlapping segments of the
operon (Fig. 2). pED3306 (22) consists of pBR322 containing
a 10-kb HindIII fragment (D fragment) with
xylUWCMA (31) and the adjacent upstream region.
pRL4 (kindly provided by S. Harayama, Kamaishi City, Japan) contains a
7.1-kb BamHI fragment generated upon insertion of
Tn5 into the TOL plasmid downstream of the upper operon.
This fragment includes the xylMABN sequences along with an
unrelated segment of Tn5. During early attempts to splice
different portions of the upper operon, it was noticed that subcloning
of a 5.8-kb XbaI-ClaI segment from pRL4 spanning
xylMABN was impossible in a multicopy plasmid. To overcome
the problem, we used a monocopy plasmid vector with an 
element
(9) in front of the cloned DNA to prevent any expression of
the inserted sequences. The specialized vector pCK02 is a derivative of
the low-copy-number, chloramphenicol-resistant vector pCK01 (10), in which a 2.0-kb -EcoRI element from
pHP45 (9) has been inserted into the EcoRI
site to stop any transcription arising from the lac promoter
present in the plasmid. pCK02 was cleaved with XbaI and
AccI and ligated to the 5.8-kb
XbaI-ClaI fragment of pRL4 containing
xylMABN. The resulting plasmid (pCK03; Fig. 2) was then
cleaved with XmaI and XbaI (thereby releasing the element) to receive a 3.3-kb XmaI-XbaI
fragment of pED3306 containing the Pu promoter and the
downstream genes xylUWC (31). This reconstructed the entire upper TOL pathway downstream of its native promoter, Pu, in plasmid pCK04. Two additional restriction sites,
KpnI and AvrII, were then created at the 5' end
of the operon sequence by insertion of a linker at the single
XmaI site of the plasmid, thus producing plasmid pCK04AK. In
addition, a 2.4-kb DNA segment spanning the regulatory gene
xylR was prepared as a KpnI fragment by adding
KpnI linkers to the ends of the HpaI fragment
released upon digestion of plasmid pTK19 (11) with this
enzyme. The KpnI segment was then inserted at the single
KpnI site of pCK04AK, giving rise to pCK04AxylR.
This plasmid carries a ca. 12-kb NotI insert (named
hereafter the upp TOL catabolic segment; Fig. 2) with the
entire TOL upper pathway, along with its cognate regulatory gene,
xylR.
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FIG. 2.
Reconstruction of the upper TOL gene cluster as a single
catabolic segment. The organization of the xyl genes of the
upper TOL operon is shown at the top, aligned with the restriction map
of the portion of the pWW0 plasmid involved (14, 31). Genes
xylUW and xylN are not required for degradation
of toluene, but they form part of the same transcriptional unit. The
location and orientation of the toluene-responsive promoter of the
system, Pu, are also indicated. Pu becomes
activated by the XylR protein (which maps at a distant position in the
pWW0 plasmid; 21) when cells encounter toluene or xylenes in the medium
(1, 21). The DNA inserts of plasmids pED3306 and pRL4 used
as the source of the xyl genes are indicated. The
organization of the constructions that preceded the reassembly of the
operon along with the xylR gene in pCK04AxylR is
shown (see the text for an explanation). The various segments were
cloned between the NotI sites of vector pCK01. This is a
Cmr plasmid with a pSC101 replicon (i.e., monocopy) and an
 -lac fragment in which the polylinker of pUC18 is flanked
by NotI sites. This allows the excision of the cloned DNA
segments as NotI fragments for further insertion in the
single NotI site of the transposon vector of pJMS11 (see
Fig. 3). Restriction sites: N, NotI;
E, EcoRI; Xm, XmaI;
Xb, XbaI; H, HindIII;
C, ClaI; A, AccI;
Av, AvrII.
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The functionality of every enzymatic step of the pathway was verified
in vivo as follows. Presentation of indole to Escherichia coli cells bearing pCK04AxylR (Fig. 2) in the presence
of 3-methylbenzyl alcohol resulted in accumulation of a dark-blue
precipitate (indigo), which revealed the activity of the enzyme xylene
monooxygenase (22), which is encoded by xylMA
(Fig. 1). When the same strain was exposed to vapors of 3-methylbenzyl
alcohol, we observed the transient formation of a product which
migrated the same as 3-methylbenzaldehyde in a high-performance liquid
chromatography system (data not shown), thus indicating the
functionality of the second enzyme of the pathway, benzyl alcohol
dehydrogenase (encoded by xylB). Finally, pCK04AxylR was cotransformed along with the compatible
xylS+ plasmid pKT570 (20, 21) in the
specialized strain E. coli CC118Pm-lacZ, which
bears a chromosomal lacZ fusion to the
m-toluate/XylS-responsive promoter Pm
(17). In the presence of 3-methylbenzyl alcohol, this strain
accumulated high levels of 
-galactosidase, i.e., 
5,000 Miller
units (23), which is equivalent to 15-fold induction. This
was evidence of the conversion of the aromatic substrate into
m-toluate by the enzyme benzaldehyde dehydrogenase (encoded by xylC) because m-toluate activates XylS, which
in turn activates the chromosomal Pm-lacZ fusion of the host
E. coli strain (21). These observations indicated
that the 12-kb upp TOL catabolic segment contains all of the
activities of the TOL upper pathway required for the bioconversions of
interest (31). Although the products of the xylUW
(31) and xylN (14) genes do not have a
known role in the degradation of toluene, they were kept in their
original configuration within the upper TOL operon to ensure reliable
performance of the system.
Insertion of the hybrid mini-Tn5 [upp
TOL] transposon into P. putida and excision of the
selection marker.
Cloning of the NotI insert of
pCK04AxylR spanning the upper TOL operon plus
xylR (Fig. 2) into the unique NotI site of pJMS11 (Fig. 3) gave rise to plasmid pCK05, the
organization of which is sketched in Fig.
4. The minitransposon assembled in pJMS11 contains a DNA segment encoding a selection marker (npt,
which encodes Kmr) and a visual marker (xylE,
encoding catechol 2,3-dioxygenase). The latter causes colonies to
become yellow upon spraying with catechol (C23O+
phenotype). This xylE/npt marker segment is flanked by two
tandem res sequences recruited from the multimer resolution
system (mrs) of plasmid RP4 (8, 12, 13, 18). The
mrs system is a site-specific recombination mechanism
involving parA, a site-specific resolvase that promotes the
deletion of any supercoiled DNA placed between two directly oriented
res sites. The result of this process is excision of the
intervening nucleotide sequence when cells express the cognate
resolvase encoded by gene parA (8, 18).
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FIG. 3.
Organization of pJMS11. This plasmid is the delivery
vector for the minitransposon shown at the top and was constructed by
deleting the NotI insert of pJMS10 (18) and
further religation to recover a unique NotI cloning site.
The suicide donation system (bottom) is that of pUT (7) and
includes the Tn5 transposase gene devoid of NotI
sites (tnp*), and Apr selection marker
(bla), an origin of transfer for RP4-mediated mobilization
(oriT), and the origin of replication of plasmid R6K, which
is dependent on the  protein encoded by the pir gene
carried by specialized  pir E. coli hosts. At the top are
the elements included in the mini-Tn5 transposon vector
portion of the plasmid. Besides the single NotI site used
for cloning of heterologous DNA segments, the predominant feature of
this plasmid is the presence of a DNA segment carrying the
xylE (C23O) and npt (Kmr) genes
flanked by tandem res sites. The arrow-shaped box in front
of the npt gene indicates the orientation of its promoter
(P). Similarly, the triangles in front of the xylE and
npt boxes indicate the orientation of their structural
genes. The res sequences are represented by the shaded
boxes. Cloning of a DNA segment at the single NotI site of
the plasmid gives rise to a hybrid mobile element flanked by the 19-bp
I and O ends of Tn5. Restriction sites: E,
EcoRI; Xm, XmaI; H,
HindIII; Sf, SfiI; Av,
AvrII.
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FIG. 4.
Insertion of the upp TOL catabolic segment
into the chromosome of P. putida KT2442 and deletion of
selection markers. The organization of pCK05, the delivery plasmid for
the mobile element mini-Tn5 [upp TOL], is shown
at the top (the vector part is not to scale). It includes all of the
features of pJMS11 (Fig. 3) plus the NotI insert of
pCK04AxylR (upp TOL segment) within the
boundaries of the I and O ends of Tn5. This plasmid was
mobilized to P. putida KT2442, and the insertion of the
mini-Tn5 [upp TOL] transposon was selected with
kanamycin and verified upon spraying of the exconjugants with catechol.
In a subsequent step, the Kmr/xylE markers were
deleted from P. putida KT2442::mini-Tn5
[upp TOL] by mobilization of the suicide plasmid pJMSB8
(which bears the parA sequence downstream of
lacI/Plac). The final result is stable inheritance of the
remainder of the hybrid transposon, i.e., mini-Tn5
[upp TOL]  [Kmr/xylE].
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|
On this basis, pCK05 became the delivery plasmid for the mobile element
mini-Tn5 [upp TOL]. Insertion of this
minitransposon into the chromosome of P. putida KT2442 was
carried out by following published protocols (7, 18).
Insertions were selected through resistance to kanamycin and verified
upon spraying of the exconjugants with catechol. The insertion
frequency of mini-Tn5 [upp TOL], defined as the
ratio of the number of inserts to the number of recipients
(7), was 10
5, a value within the range of
other mini-Tn5 transposons carrying smaller DNA segments
(16). Of 50 Kmr and piperacillin-sensitive
yellow colonies tested, 30 were able to grow on toluene as the only
carbon source (Fig. 5).
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FIG. 5.
Phenotypes of P. putida KT2442 inserted with
the upp TOL catabolic segment. Shown is the result of
patching three related P. putida KT2442 strains in minimal
medium with the carbon source and the antibiotic indicated in each case
and spraying them with 1% catechol after growth. Strain 1 is P. putida KT2442 without any insertion. It can grow only in the
medium with citrate and remains white. Strain 2 is P. putida
KT2442 inserted with the complex transposon mini-Tn5
[upp TOL]. The presence of the xylE and
npt markers in the transposon (Fig. 4) causes the patch to
become yellow (C23O+) and to grow in the presence of
kanamycin (Km), as well as on toluene, as the only carbon source.
Strain 3 is a derivative of strain 2 in which the xylE/npt
portion of mini-Tn5 [upp TOL] has been deleted
in vivo. Where indicated, toluene was provided as a saturating vapor
phase.
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Sensitivity to piperacillin ensured the occurrence of authentic
transposition events (7), since it rules out the integration of the whole delivery plasmid into the P. putida chromosome.
The 30 clones with the phenotype caused by insertions of
mini-Tn5 [upp TOL] were separately subjected to
triparental mating (7) with E. coli CC118(pJMSB8)
and the helper strain E. coli HB101(RK600). The pJMSB8
plasmid is used for transient expression of the RP4 resolvase gene
parA (18), whereas RK600 assists in the conjugal transfer of pJMSB8 from E. coli to Pseudomonas
(7). To achieve transient expression of parA,
overnight cultures of E. coli(pJMSB8), P. putida KT2442:: mini-Tn5
[upp TOL], and E. coli HB101(RK600) were
separately washed, mixed at 10:1:10, respectively, and then spotted
onto the surface of a Luria broth plate. After 4 to 8 h of
incubation at 30°C, spots were streaked onto the surface of M9
minimal medium plates with 10 mM citrate as the carbon source (23) but without antibiotics. This allowed growth of
Pseudomonas but not of the E. coli strains
present in the mating, which cannot use citrate as a carbon source. The
plates were then sprayed with catechol to reveal the loss of the
xylE marker (along with Kmr). With this
procedure, >80% of the colonies had the phenotype anticipated for
those losing the xylE/npt marker segment of the transposon
while retaining the ability to grow on toluene as the only carbon
source (Fig. 5). Further analysis of the strains by Southern blotting
(26) and PCR verified the presence of the entire
upp TOL element in the chromosome of P. putida
KT2442 and the loss of the xylE/npt cassette (data not
shown).
Characteristics of P. putida derivatives bearing the
mini-Tn5 [upp TOL]
[Kmr/xylE] insert.
The strains
resulting from the insertion of mini-Tn5 [upp
TOL] and subsequent removal of the Kmr/xylE
segment inherited a new phenotype (growth on toluene due to the
acquisition of the upp TOL segment) with no other additional markers. Although the concepts for the genetic manipulations were laid
out before (18), this work demonstrates their practical utility and its extension in the design of novel bacterial
biocatalysts. The recruitment of a DNA fragment to the chromosome of
P. putida through this procedure resembles the natural
mechanisms of insertion and deletion of DNA that occur during natural
adaptation processes (15, 27) to such an extent that the
constructed strains can be considered quasi-natural. In fact, the final
product of the manipulations described here is somewhat reminiscent of
that of the naturally occurring genetic event designated
"transposition without transposase" by Rappleye and Roth
(24). Although in our case, the transposase of
Tn5 is used to effect the insertion of the DNA segment, the
tnp gene is not inherited by the resulting strain (7,
16). This makes it extremely unlikely that the insertions can be
moved again. In this respect, the new DNA segment(s) added to the
genome of the recipient strain (in our case, the upper TOL pathway) can
be even more stable and predictable than many catabolic genes of
natural Pseudomonas isolates, the DNA sequences of which are
systematically subjected to rearrangements (30). In
contrast, no loss of the hybrid mini-Tn5 insertion was
detected after 1,000 generations (data not shown).
The strains bearing mini-Tn5 [upp TOL]
[Kmr/xylE] grew on toluene as the only
carbon source (Fig. 5). The rationale of such a phenotype is the
conversion of toluene to benzoate, mediated by the upper TOL enzymes
encoded and expressed by the insertion and the further metabolism of
benzoate through the housekeeping ortho-ring cleavage
pathway of the catechol intermediate (Fig. 1). This pathway for
degradation of toluene, which avoids a meta-cleavage step,
is infrequent in natural isolates and will permit us to tackle the
biodegradation of difficult chemical species such as chlorotoluenes
(1, 2, 19). In a different context, the separate expression
of the entire upper TOL pathway away from the meta operon
should allow bioconversion of an array of substituted toluenes to the
corresponding acids (2, 3).
| |
ACKNOWLEDGMENTS |
We are indebted to S. Molin, C. Sternberg, and S. Harayama for
facilitating some of the materials and ideas exploited in this work.
K. N. Timmis and D. Pieper are also gratefully acknowledged for
their support.
This work was funded by contract ENV4-CT95-0141 (ENVIRONMENT) with the
European Union and by grant AMB94-1038-CO2-02 from the Spanish
Comisión Interministerial de Ciencia y Tecnología.
S.P. and J.M.S.-R. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro Nacional
de BiotecnologíaCSIC, Campus de Cantoblanco, Madrid 28049, Spain. Phone: 341-585 4536. Fax: 341-585 4506.
Present address: Institut für Biotechnologie, ETH,
Hönggerberg, HPT D73 8093 Zürich, Switzerland.
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Appl Environ Microbiol, February 1998, p. 748-751, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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