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Applied and Environmental Microbiology, September 2003, p. 5120-5127, Vol. 69, No. 9
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.9.5120-5127.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain,1 Central Research and Development, DuPont Experimental Station, Wilmington, Delaware 19880-03282
Received 6 February 2003/ Accepted 18 June 2003
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ABSTRACT |
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and ß subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1E
todC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The P. putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant. This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EtodCpobA, that was unable to use either toluene or 4-HBA as a carbon source. The tmo and pcu genes from P. mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1EtodCpobA. Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24. |
INTRODUCTION |
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4-HBA is an added-value compound used in the synthesis of paraben and methylparaben, which are p-hydroxybenzoic acid alkylic ester derivatives that are of great interest for the synthesis of liquid glass (9) and as antimicrobial agents (23, 24). The potential to use 4-HBA as a carbon source is widespread in gram-negative soil bacteria, and it is not a unique property of species that have the T4MO pathway as the toluene catabolic pathway. 4-HBA is hydroxylated to 3,4-dihydroxybenzoate by the activity of the pobA product. 3,4-Dihydroxybenzoate undergoes ortho cleavage and is catabolized via the ß-ketoadipate pathway (7).
The high tolerance of DOT-T1E to organic solvents makes this strain a good candidate for biotransformation and particularly for conversion of highly toxic substrates, such as toluene, into 4-HBA. Strain DOT-T1E tolerates up to 18 g of 4-HBA, the product of the biotransformation, per liter. This value increases to 30 g/liter when cells are preinduced with 4-HBA (18). P. mendocina KR1 is considerably less tolerant to toluene and 4-HBA than P. putida DOT-T1E; it tolerates less than 0.1% (vol/vol) toluene and 8.5 g of 4-HBA per liter (unpublished observations), and there is subsequent loss of viability during the process (Ben-Bassat, unpublished results).
The rationale behind this study was to recruit the appropriate enzymatic machinery for biotransformation of toluene into 4-HBA in the solvent-resistant DOT-T1E strain. To avoid misrouting of toluene and 4-HBA through the Tod and ß-ketoadipate pathways, respectively, the first steps of these routes were inactivated in this strain. The double mutant was then equipped with the tmo and pcu genes from P. mendocina KR1 so that toluene was efficiently converted into 4-HBA, which remained in the liquid phase.
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MATERIALS AND METHODS |
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todCpobA double mutant, which is not able to use toluene and 4-HBA as carbon sources, was generated in this study.
Mobilizable but not self-transmissible plasmids were transferred to recipient hosts by using triparental mating involving Escherichia coli HB101 bearing the helper plasmid pRK600. Plasmids with the replication origin ori R6K were maintained in E. coli CC118 
pir (8). Plasmids used and constructed in this study are listed in Table 1. E. coli and Pseudomonas strains were cultivated in Luria-Bertani (LB) medium or modified M9 minimal medium supplemented with 25 mM glucose and/or toluene in the gas phase as a carbon source (13), and they were incubated at 37 and 30°C, respectively. Competent E. coli DH5F' cells were used in transformation experiments (27). Electrotransformed cells were incubated in SOC medium for 1 to 2 h to allow expression of the acquired antibiotic resistance markers. Antibiotics were added as required to the media at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 30 µg/ml; gentamicin, 10 µg/ml for E. coli strains and 100 µg/ml for pseudomonads; kanamycin, 25 µg/ml; streptomycin, 50 to 150 µg/ml; piperacillin, 90 µg/ml; tetracycline, 10 µg/ml; and trimethropim, 75 µg/ml. Potassium tellurite was used at a concentration of 15 µg/ml.
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Plasmid construction.
pUC18NB-H is a pUC18Not derivative that lacks part of the polylinker of pUC18Not (8). This plasmid was generated after double digestion of pUC18Not with BamHI and HindIII and filling in of the protruding ends with the Klenow enzyme and deoxynucleoside triphosphates.
Plasmid pMIR22 is shown in Fig. 2. The entire tod operon was available in two plasmids: plasmid pT1-4, in which the todXF genes had been cloned; and plasmid pT1-125, containing the todC1C2BADEGIH gene cluster and the todST regulatory genes (13). Plasmid pMIR21 was obtained like pMIR22, but it had a 3-kb BamHI/HindIII fragment from pUT/Tel (22) containing the kilA telAB genes instead of the kanamycin resistance cassette. Plasmid pMIR29 was obtained like pMIR30 except that subcloning was with the NotI fragment of pMIR21 with the todF'todC1::kilAtelAB::'todC2 region.
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/Sm cassette, was incorporated at the unique NotI site present in the AT-2 transposon of pMIR27 to create plasmid pMIR31.
Electrotransformation of P. putida todCkm cells with pMIR31.
We used the procedure described by Enderle and Farwell (3) to electrotransform P. putida cells. Cells in 0.2-cm cuvettes were subjected to a high-voltage pulse (3,000 V) for 5 ms by using a MicroPulser electroporation apparatus (Bio-Rad). Cells were subsequently incubated in 1 ml of SOC medium for 2 h at 30°C and then harvested by centrifugation (13,000 x g for 2 min in a microcentrifuge), and the pellet was incubated overnight at the same temperature on a filter placed on an LB agar plate. The cells were finally resuspended in 1 ml of LB medium and plated on LB agar supplemented with kanamycin and streptomycin (25 and 150 µg/ml, respectively).
Quantitative detection of 4-HBA in the culture supernatant by high-performance liquid chromatography.
4-HBA that accumulated in culture supernatants was analyzed by high-performance liquid chromatography by using a Hewlett-Packard model 1050 chromatograph equipped with a diode array detector and a 5-mm C18RP column (UltraCarb C30 Phenomenex; 15 cm by 4.6 mm). The column was first washed with a mixture of acetonitrile and a solution of 1% (vol/vol) acetic acid in water (2:8, vol/vol) for 2 min. A linear gradient was then applied to reach 60% (vol/vol) acetonitrile over 15 min. The flow rate was kept constant at 1 ml/min, and the detector was set at 230 and 254 nm to detect aromatic compounds.
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RESULTS |
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todC1::km::'todC2).
Plasmids pMIR29 and pMIR30 are derivatives of the suicide vector pKNG101 (10), whose construction is described above (Fig. 2). These plasmids carry the insertional deletions todF'todC1::km::'todC2 and todF'todC1::kilAtelAB::'todC2, respectively, and were used as delivery plasmids to replace the wild-type toluene dioxygenase in the chromosome of DOT-T1E with the deleted version by homologous recombination. Details concerning the methods used for mobilization of these suicide plasmids, selection of merodiploid strains, and selection of mutants upon allelic exchange have been described previously (19). Two mutants unable to use toluene as a carbon source were isolated and designated todCkm and todCtel, respectively. Deletion of the todC1 gene in the chromosome of the toluene-minus isolates was confirmed by PCR with specific primers and by Southern blotting (data not shown). The stabilities of the two mutants were tested and were found to be identical: (i) after 90 generations of growth in LB medium under nonselective conditions (with no antibiotics), 100% of the cells were resistant either to kanamycin or to tellurite and were unable to grow in toluene; (ii) no growth was observed after 1 week of incubation in M9 liquid minimal media with toluene as the sole C source in the absence of selective pressure for the markers; and (iii) the reversion rate, determined by measuring reacquisition of the ability to grow on toluene for both clones tested by the drop plating technique, was less than 10-9.
Analysis of the sequence in the P. putida pobA-pobR region.
Plasmid pMIR27 was generated by in vitro random transposition with the AT-2 transposon as described above. This plasmid had the transposon inserted at the 5' end of the pobA gene (between positions 123 and 124 after the ATG), with consequent interruption of the gene. The whole pobA gene and part of the pobR gene were identified in pMIR27. As in other pseudomonads, the two genes were transcribed divergently. The complete sequence of the pobA gene revealed that it consists of a 1,188-nucleotide open reading frame at positions 82783 to 83970 in the genome of P. putida KT2440 (accession number AE015451). The clone contained part of the pobR gene, whose putative start codon was located 173 nucleotides upstream of the ATG start site of the pobA gene, in the antiparallel strand. The overall levels of identity between the P. putida pobA gene and the P. mendocina pobA1 and pobA2 genes were 77 and 79%, respectively. The level of identity between the two P. mendocina pobA genes was 84%. At the protein level, the overall level of identity between the PobA1 and PobA2 proteins was about 90%, whereas the deduced amino acid sequence encoded by the P. putida pobA gene exhibited 83 and 84% identity with the P. mendocina PobA1 and PobA2 proteins, respectively.
Generation of 4-HBA-minus derivative of P. putida todCkm (double mutant todCpobA).
Plasmid pMIR31 was used as a delivery system for gene replacement by homologous recombination of the wild-type pobA allele by an inactivated copy. P. putida todCkm cells were eletrotransformed with pMIR31 as described above, and the resulting Kmr Smr clones were tested for the ability to grow on 4-HBA and for Pipr, the marker of pMIR31. One clone which was unable to use 4-HBA as a C source and which was Pips was isolated. The successful allelic exchange of the wild-type pobA gene for the inactivated copy in the todCkm/pobA::Sm double mutant was confirmed by Southern blotting (data not shown). The inactivated pobA gene was stable, and no revertants able to grow on 4-HBA were detected after 1 week of incubation in the presence of the hydroxylated carboxylic acid.
Stable transfer of mini-Tn5Tctmo/pcu into the chromosome of DOT-T1EtodCpobA.
The P. putida DOT-T1EtodCpobA mutant was mated with E. coli CC118pir(pMIR44) in the presence of the helper organism E. coli(pRK600), and exconjugants of P. putida were selected on LB medium supplemented with kanamycin, streptomycin, and tetracycline at a rate of 5 x 10-8 exconjugant per recipient. From the exconjugants, six clones that were able to grow in minimal medium with glucose as fast as the parental strain were isolated. All of these exconjugants failed to grow with either toluene or 4-HBA as the sole carbon source because of the pobA mutation. The stability of each of the markers was tested. The six strains were cultivated on LB medium without antibiotics for about 100 generations. After serial dilutions were spread on LB medium, the results confirmed that 100% of the cells retained all antibiotic markers. The transposon was detected by Southern blotting in the chromosome of five of the isolates at different positions (Fig. 3B).
Effect of toluene on the growth and survival of P. putida DOT-T1E wild-type and mutant strains.
The sensitivity of bacterial cells to toluene, the substrate of the biotransformation reaction, might be critical during the process. The growth characteristics of the wild-type DOT-T1E strain, the null todC mutant strain, the double mutant todCpobA strain, and six independent exconjugants of the double mutant strain with mini-Tn5Tctmo/pcu were compared in LB medium and in glucose-supplemented M9 minimal medium with the appropriate antibiotics. All the strains exhibited similar growth curves in LB medium in the absence of toluene (data not shown). However, when toluene was supplied in the gas phase, the double todCpobA mutant exhibited the same growth profile as the wild-type strain, whereas all the strains bearing mini-Tn5Tctmo/pcu grew more slowly than the parental strains (Fig. 4A shows the growth of two 4-HBA producer clones as an example). This observation might have been due to the energy requirements for the biotransformation. In minimal medium in the absence of toluene, similar growth was observed for all of the strains; however, in the presence of toluene, the growth rates of the todCpobA double mutant and its producer derivatives were lower (the doubling time decreased from 65 to 200 min) (Fig. 4B). Toluene also had a negative effect on the highest cell density reached by the mutants.
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todCpobA derivatives carrying mini-Tn5Tctmo/pcu in the chromosome.todCpobA derivatives and accumulated in the culture medium. In order to determine the effect of the insertion site of the minitransposon on the efficiency of 4-HBA, a screening experiment with a pool of independent exconjugants was carried out to analyze possible differences in 4-HBA production. The production of 4-HBA was studied with resting cells at a high cell density (optical density at 660 nm, about 10 U; 1010 CFU/ml) in glucose-supplemented M9 minimal medium (Fig. 5A). Cell density was constant in the experiment (data not shown), whereas production of 4-HBA exhibited a lag in all cases, which varied from 3 h in clon-24 to more than 24 h in clon-1-5A. The concentration of 4-HBA was as high as 10.5 mM in the most proficient strain, clon-24, which exhibited a rate of production of 0.4 mM/h during the interval when lineal production occurred. The lowest concentration detected, less than 4 mM, was the concentration observed with clon-17. P. mendocina KR-303 was used as a 4-HBA-producing control strain (Ben-Bassat et al., U.S. patent application 20020151003). The kinetics of 4-HBA accumulation in this strain are shown in Fig. 5B. The time required for detection of the hydroxylated aromatic acid in the supernatant of this strain was similar to the lowest lag time determined for any of the P. putida producers, although an unexpected decrease in the amount accumulated was observed after 30 h of incubation. Production of 4-HBA by P. putida DOT-T1E derivatives was also analyzed at a lower cell density (turbidity at 660 nm, 1 U) along the growth curve. Cells in the early stationary phase were removed from exhausted medium and amended with fresh medium. Under these conditions, growth of the cells at the expense of glucose was observed with concomitant 4-HBA production. The production by two clones is shown in Fig. 6. The time required for detection of certain amounts of 4-HBA was similar to the time required with resting cells.
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DISCUSSION |
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Biotransformation of toluene to 4-HBA occurs naturally in P. mendocina KR1 through the T4MO pathway, which is responsible for the hydroxylation of toluene to p-cresol and the subsequent oxidation of p-cresol to 4-HBA by the enzymes encoded by the p-cresol utilization genes (pcu) (28) (Fig. 1, pathway B). In the wild type, 4-HBA is used as a carbon source by action of the p-hydroxybenzoate hydroxylase and the subsequent ß-ketoadipate pathway, so that the amount of hydroxylated acid in the supernatant is undetectable (data not shown). However, in strain KR1-303, a pobA1 pobA2 double mutant (Ben-Bassat et al., U.S. patent application 20020151003), accumulation of up to 6.3 mM 4-HBA was detected after 10 h in batch cultures, and there was a slight increase to 7.5 mM after 30 h of incubation (Fig. 5B).
The mutant DOT-T1EtodC was as tolerant as the wild type to a sudden shock of toluene and 4-HBA, which were added as reported previously (5, 16). The independence of solvent tolerance and biodegradation capacity was not unexpected. Although metabolism of toxic chemicals can help reduce their toxicity, this mechanism should contribute considerably less than other major mechanisms as efflux pumps in protecting cell viability (15). Like the DOT-T1EtodC single mutant, the todC pobA double mutant was as tolerant as the wild type to a sudden shock of toluene and 4-HBA (data not shown), despite the fact that it did not use any of the compounds as a carbon source.
The tmo and pcu gene clusters in mini-Tn5Tctmopcu (Fig. 3) were responsible for the production of 4-HBA by resting cells of the todC pobA double mutant, and different amounts of the compound were detected for independent clones (range, 3.5 to 10.5 mM) (Fig. 5A). In the clones with the highest rates of 4-HBA production, DOT-T1E-21 and DOT-T1E-24 (0.38 and 0.4 mM/h, respectively), the concentration reached a maximum after 28 to 30 h. Addition of a carbon source (glucose) to the samples did not restore the production (data not shown), in agreement with the previous observation that the rate-limiting step in 4-HBA production is T4MO, which has a half-life of 28 h (12). The fact that 4-HBA production from glucose has been described for a series of recombinant E. coli strains should be taken into account; this production occurs through a complex process that involves up to eight steps after phosphoenolpyruvate (1), compared with the four steps in our system. In E. coli in fermentors, the level of 4-HBA accumulation was about one-third (12 g/liter) the level obtained under similar conditions with Pseudomonas (6).
Expression of the tmo promoter in P. mendocina KR1 requires a two-component signal transduction system designated tmoS/tmoT (20). In DOT-T1E, as well as in the 4-HBA-producing derivatives generated in this study, there are homologues of tmoS/tmoT (i.e., todST, the two-component signal transduction system that regulates expression of the tod operon) (13). In a recent study, activation of tmo genes by the TodST system was confirmed (20), and this cross-activation, together with the robustness of DOT-T1E, made this strain a good candidate for biotransformation of toluene into 4-HBA.
A pobA mutant derivative of P. putida KT2440 was generated in this study. Mini-Tn5Tctmopcu was recruited into this mutant, and after a pool of exconjugants with the transposon at different positions in the chromosome was screened, as done previously with the todCpobA derivatives, no 4-HBA production from toluene was detected. Since there are no homologues of the TmoST regulator in strain KT2440 (20), the lack of induction of the tmo genes was most probably responsible for the failure of biotransformation in this strain. In a previous study with a KT2440 pobA strain, which carried tmo and pcu gene clusters in two transposons inserted into the chromosome, production of 4-HBA from toluene was reported (12). In that strain, which did not have tmoST genes, toluene-dependent activation from the tmoX promoter was not feasible. Instead, inducible promoters in the presence of appropriate activators were used.
Although we did not observe any decrease in the accumulation of 4-HBA with the producer derivatives of todCpobA, such a decrease was observed with P. mendocina KR1 clon-303 (Fig. 5B). To our knowledge, catabolic turnover should not have been the reason for the decrease in the amount of 4-HBA observed since both pobA genes present in P. mendocina KR1-303 had been knocked out and the possibility of there being a third pobA gene was eliminated by hybridization (data not shown). However, 4-HBA could have served as a substrate for enzymes involved in catabolism of aromatic compounds other than p-hydroxybenzoate hydroxylase. In fact, evidence of 4-HBA metabolism via alternative pathways has been obtained for the wild-type strain DOT-T1E (Ramos-González, unpublished data).
Toluene in the gas phase slowed the growth of producing strains (Fig. 4). This effect could have been a consequence of the extra energy demand due to transformation of toluene. However, the same result was observed with the nonproducing todC pobA strain. The effect of toluene was not due to a decrease in tolerance to toluene or 4-HBA since all strains were as resistant as the wild-type and todCpobA strains (data not shown). The negative effect of toluene was not life-threatening for the exponentially growing cells that accumulated 4-HBA in the supernatant at rates ranging from 0.23 to 0.5 mM/h (Fig. 6). The strain that accumulated larger amounts of 4-HBA was clone DOT-T1E-24 (which accumulated up to 12 mM).
In short, our results indicate that inactivation of the tod and pobA genes of P. putida DOT-T1E and incorporation of the tmo and pcu genes produce suitable, highly stable clones that can be used for biotransformation of toluene into 4-HBA, a compound of great industrial interest.
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ACKNOWLEDGMENTS |
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We greatly thank Anthony A. Gatenby for supplying plasmid pMC4. We also thank Ana Hurtado for DNA sequencing, Carmen Lorente and M. Mar Fandila for secretarial assistance, and M. Espinosa for his kind help with improving the language.
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FOOTNOTES |
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REFERENCES |
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4.5-kb EcoRI/XcaI fragment of pT1-125, which carried genes todC1 to todD, was subcloned at the EcoRI/SmaI sites of pUC18NB-H, generating plasmid pMIR17. (C) The 1.8-kb SspI/EcoRI fragment of pT1-4, containing the todXF genes, was subcloned at the EcoRI site of pMIR17 to obtain plasmid pMIR20 (the unique NotI site present in the SspI/EcoRI fragment was removed before cloning). (D) Most of the 3' half of todF, the entire todC1 gene, and the 5' end of todC2 were removed from pMIR20 as a 2.2-kb BamHI/HindIII fragment. (E) Insertion of a 2.2-kb 
), the 
), the 
), and two derivatives of 
) and DOT-T1E-1-10a ([ast]). (B) Same as panel A except that glucose-supplemented M9 minimal medium was used instead of LB medium. The appropriate antibiotics were added to the cultures. OD 660 nm, optical density at 660 nm.
