Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain

Abstract

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.

Substituted picolinic acids are used as feedstocks for the synthesis of a variety of pharmaceuticals, agricultural chemicals, and dyes (1, 12). Chemical synthesis involves several steps and the use of harsh reagents, and low yields of the desired products are obtained. Several strategies for using enzymes to catalyze reactions leading to the synthesis of picolinic acid have been described. The first reports (3, 29) described production of 2-hydroxymuconic semialdehyde from catechol catalyzed by dioxygenase enzymes from two strains of gram-negative bacteria. Treatment of the 2-hydroxymuconic semialdehyde with ammonium ions led to the formation of picolinic acid. This reaction has been the basis for a number of subsequent papers (1) and patents (11, 12) describing the synthesis of substituted picolinic acids. Recently, a related strategy involving cleavage of the ring of substituted 2-aminophenols by aminophenol dioxygenase from Pseudomonas pseudoalcaligenes strain JS 45 was described (16). The resultant ring fission product rearranges spontaneously to the corresponding picolinic acid. The techniques described above have been used for synthesis of relatively simple substituted picolinic acids. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid]. The potential reactivity of 6-phenylacetylene picolinate as a cross-linker or a dienophile makes it a good candidate to replace phenylacetylenes as endcapping agents for high-performance thermosetting polymers for aerospace applications (17) and electronic packaging (26). The strong electron-withdrawing properties of the pyridine moiety are predicted to allow lower reaction temperatures and shorter cure times during Diels-Alder-type polymerization. The properties of the molecule are only predicted because to our knowledge, it has not been synthesized previously.

We describe here a screening procedure to identify toluene-degrading strains that have the ability to catalyze the three reactions involved in conversion of diphenylacetylene (DPA) to the meta ring fission product, the abiotic conversion of the ring fission product to 6-phenylacetylene picolinic acid, and development of a system to sustain the conversion activity by the presence of toluene in spite of the fact that toluene is a competitive inhibitor of the enzymes. The systems developed to accomplish the synthesis described above have the potential to use the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation in order to produce novel compounds.

(A preliminary account of this work has been presented previously [S. F. Nishino, J. C. Spain, W. A. Duetz, and B. Witholt, Abstr. Am. Soc. Microbiol. Conf. Biodegrad. Biotransform. Biocatalysis, abstr. 83, p. 50, 2001].)

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The 139 toluene-degrading strains screened for the ability to transform DPA were isolated from (i) topsoil samples from different polluted sites in The Netherlands (91 strains) and (ii) soil, water, and sewage sludge samples from various sites in Switzerland (48 strains). Pseudomonas putida strain F1 and Escherichia coli JM109(pDTG603) were kindly provided by David Gibson (University of Iowa). The growth medium was Stanier's minimal medium (MSB) (28) supplemented with toluene as indicated below. Shake flask cultures were grown in 250-ml baffle flasks containing 100 ml of MSB, and toluene was provided in the headspace as described previously (14). All cultures were grown at 30°C and pH 7.0 with shaking at 200 rpm.

Larger cultures were grown in a 2-liter New Brunswick Multigen fermentor or in a 14-liter New Brunswick Multiferm fermentor. Toluene was pumped into each fermentor via a syringe pump at the flow rates indicated below.

Screening procedure.

The toluene-degrading strains were grown on toluene vapor in microplates and screened for the ability to transform DPA as follows. Multiple strains stored in a single 96-well microtiter plate at −80°C were sampled simultaneously (without thawing) with a spring-loaded 96-pin replicator (Kuhner, Basel, Switzerland) as previously described (6). The samples were transferred to a sterile microtiter plate in which each well (working volume, 350 μl) was filled with 180 μl of a solidified (2% agar) mineral medium without any carbon source. The inoculated microtiter plate (with 2 mm between the lid and the wells to allow gas diffusion) was placed in a desiccator containing a beaker with toluene dissolved in hexadecane (10%, vol/vol). After 7 days of growth at the ambient temperature (22 to 25°C), the cells were harvested as follows. First, 110 μl of phosphate buffer (50 mM, pH 7.0) was added to each well. Repeated lateral movement of the spring-loaded replicator in the wells resulted in suspension of a large part of the cell mass in the buffer. The suspensions were transferred to a microtiter plate with 0.5-ml conical wells (Maxi-plaque; Polylabo, Geneva, Switzerland). The plate was centrifuged for 15 min at 2,250 × g, and the supernatant was discarded. The cells were suspended in 100 μl of phosphate buffer, and 2 μl of DPA (50 mM in methanol) was added to each well. The plate was closed with a sandwich cover (a pierced layer of soft silicone in combination with a rigid polypropylene plate) as described previously (6). After incubation at 25°C for 18 h with orbital shaking (300 rpm; shaking amplitude, 5 cm), the microtiter plate was centrifuged for 15 min at 2,250 × g. The supernatants (50 μl) were transferred to a half-area microtiter plate (type 3696; Costar, Corning, N.Y.) and examined visually for the appearance of colored products and by high-performance liquid chromatography (HPLC)-mass spectrometry (MS) for the accumulation of transformation products.

Analytical methods.

HPLC-MS for screening studies was performed as described previously (5). The MS was operated in the selected ion mode and set to detect masses corresponding to the molecular masses of DPA (178 Da), hydroxydiphenylacetylene (210 Da), and 2-hydroxy-6-phenylacetylene muconic semialdehyde (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]) (242 Da). HPLC-MS analysis of the purified products was performed by J. V. Johnson, Department of Chemistry, University of Florida, with an LCQ (Finnigan MAT, San Jose, Calif.) equipped with an atmospheric pressure chemical ionization (APCI) interface with the following parameters: vaporizer temperature, 250°C; capillary temperature, 175°C; sheath gas, 25; aux gas, 0; discharge current, 5 μA; discharge voltage, ∼4.5 kV; capillary voltage, 15 V (APCI); and tube lens voltage, 0 V. Analytes were separated with a Symmetry Shield RP18 column (2.1 by 150 mm; Waters, Milford, Mass.) with no guard column. The mobile phase consisted of two parts, part A (0.5% acetic acid in H₂O) and part B (0.5% acetic acid in methanol). The flow rate was 0.2 ml/min. The mobile phase was changed from 25% part A and 75% part B to 100% part B over a 10-min period.

HPLC analysis during transformation experiments was performed with a Hewlett-Packard 1040A system equipped with a diode array detector. Compounds were separated by paired ion chromatography on a Zorbax octyldecyl silane column (5 μm; inside diameter, 4 mm; length, 8 cm). The mobile phase consisted of two parts, part A (5 mM PIC A low-UV reagent [Waters brand of tetrabutylammonium hydrogen sulfate] in 30% HPLC grade methanol-70% water) and part B (5 mM PIC A low-UV reagent in 75% methanol-25% water). The flow rate was 1 ml/min. The mobile phase was changed from 100% part A to 100% part B over a 5-min period, and then 100% part B was used for 8 min. DPA and 6-phenylacetylene picolinic acid were monitored at A₂₈₀, RFP was monitored at A₄₃₀, and toluene was monitored at A₂₅₄.

Room temperature nuclear magnetic resonance (NMR) spectra were obtained with a Joel-ECX-270 NMR spectrometer with a field strength of 270 MHz for ¹H and 68 MHz for ¹³C by using the standard acquisition software program. Each sample was dissolved in CDCl₃ at a concentration ∼0.01 M.

A carbon-hydrogen-nitrogen (CHN) analysis was performed with a Perkin-Elmer model 240 analyzer.

Oxygen uptake rates were determined polarographically by using Clark-type electrodes connected to a YSI model 5300 biological oxygen monitor.

Protein content was measured by using the bicinchoninic acid protocol supplied with a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, Ill.)

Extraction and purification of products.

When biotransformations were complete, cells were removed by cross-flow filtration with a Millipore Pellicon tangential flow filter system or by centrifugation at 26,000 × g. The culture fluid was extracted with hexane to remove the remaining DPA, and the aqueous phase was passed through a C₁₈ solid-phase extraction (SPE) column (12 g; Waters). The column was washed with 25% methanol, and the RFP was eluted with 30% methanol. The methanol was removed under a vacuum, and the pH of the aqueous phase was adjusted to 3.6. Then the aqueous phase was passed through a second C₁₈ SPE column as described above for further purification. The methanol was removed under a vacuum, and the purified RFP was concentrated by freeze-drying. RFP in methanol was converted to 6-phenylacetylene picolinic acid by treatment with 8 N NH₄OH overnight at 60°C. The pH of the 6-phenylacetylene picolinic acid solution was adjusted to pH 3.6, deionized water was added to reduce the methanol content to less than 25%, and the solution was passed through a C₁₈ SPE column. The column was washed with 25% methanol, and the 6-phenylacetylene picolinic acid was eluted with 30% methanol. The fraction that eluted in 30% methanol was diluted with 25% methanol and passed through a fresh C₁₈ SPE column for further purification. The methanol was removed from the purified 6-phenylacetylene picolinate under a vacuum, and the 6-phenylacetylene picolinate was concentrated by freeze-drying. The freeze-dried product was recrystallized from hot heptane before NMR experiments and CHN analysis. The RFP could be converted to 6-phenylacetylene picolinate by direct addition of NH₄OH to the clarified culture medium, but the preliminary SPE step provided a cleaner product and greatly reduced the volume of fluid that had to be treated with NH₄OH.

RESULTS AND DISCUSSION

Initial experiments with P. putida F1 and a clone containing the todC1C2BADE genes from strain F1, E. coli JM109(pDTG603) (31), indicated that the enzymes that catalyze toluene catabolism in strain F1 do not catalyze the transformation of DPA to RFP. We therefore screened a collection of toluene-degrading strains for the ability to catalyze the transformation. Of the 139 strains examined, 37 produced a yellow, fluorescent metabolite, and HPLC-MS analysis revealed the presence of putative RFP, as indicated by the molecular ion (m/z 243) at a retention time of 1.74 min. The strain selected for subsequent experiments was identified as Acinetobacter sp. strain F4 based on a partial 16S rRNA analysis (MIDI Labs, Newark, Del.).

Optimization of transformation.

Previous work indicated that inclusion of toluene during biotransformation experiments with other toluene-degrading strains inhibits the transformation because toluene is a competitive inhibitor of the oxygenases involved (5). Shake flask experiments with strain F4 (Fig. 1) revealed that resting cells catalyzed the transformation at a considerable rate initially, but the activity declined rapidly. When toluene was included in the headspace of the flasks, the activity was sustained. These results suggested that addition of appropriate amounts of toluene might enhance transformation in larger-scale experiments if competitive inhibition could be minimized.

FIG. 1.

DPA conversion to RFP in the presence of toluene vapor (▪) and in the absence of toluene (□). A 100-ml shake flask culture was grown overnight on toluene vapor supplied in the headspace, washed, and inoculated into fresh medium containing DPA. The culture was divided in half, and one portion received toluene in the headspace.

The toluene feed rate required to support growth (final optical density at 600 nm, 10 to 30) of the biomass in a 2-liter reactor (0.65 ml h⁻¹ g [dry weight]⁻¹) inhibited the transformation of DPA (Fig. 2). When the culture had grown to an appropriate cell density, an experiment was conducted to determine the toluene feed rate that supported production of the transformation product while minimizing competitive inhibition. DPA was added to the reactor when the toluene feed rate was 0.65 ml h⁻¹ g (dry weight)⁻¹, and at intervals the toluene feed rate was decreased. Production of RFP was insignificant until the toluene feed rate reached 0.060 ml h⁻¹ g (dry weight)⁻¹. A toluene feed rate of 0.031 ml h⁻¹ g (dry weight)⁻¹ supported an optimal rate of conversion.

FIG. 2.

Transformation of DPA by stationary cells. Cells were grown in a 2-liter bioreactor, DPA was added, and the toluene feed rate was decreased stepwise until DPA transformation was maximal. OD₆₀₀, optical density at 600 nm.

Based on the results described above, the DPA conversion efficiency was determined in a subsequent experiment in the same reactor (Fig. 3A). The cells were grown to an appropriate density, and then the toluene flow rate was reduced to 0.046 ml h⁻¹ g (dry weight)⁻¹ prior to addition of DPA. Production of RFP began immediately and continued until the DPA was exhausted. The toluene feed rate was also empirically determined in a 14-liter reactor by allowing cells to grow until toluene became limiting (Fig. 3B). The toluene feed rate was then kept constant, and DPA was added to the culture. The DPA was immediately converted to RFP. The toluene feed rate was 0.052 ml h⁻¹ g (dry weight)⁻¹. The close agreement between the rates determined by the two methods suggests that use of toluene-limited cultures for transformation might be the simplest method of adjusting the toluene feed rate to the proper level. Under transformation conditions, a toluene consumption rate of 423 μmol h⁻¹ g (dry weight)⁻¹ resulted in an RFP synthesis rate of 168 μmol h⁻¹ g (dry weight)⁻¹. In comparison, during rapid growth the toluene consumption rate was 8,994 μmol h⁻¹ g (dry weight)⁻¹, assuming that there was complete uptake of the toluene in the feed.

FIG. 3.

DPA transformation at optimal toluene flow rates. The toluene flow rate was determined by extrapolating the results shown in Fig. 2 (A and C) (2-liter reactor) or by allowing the culture to reach the stationary phase due to toluene limitation (B) (14-liter reactor). DPA was added in methanol (A) and in hexane (C). The DPA concentration is the dissolved concentration, not the actual amount added. Undissolved crystals of DPA went into solution as DPA was transformed. OD₆₀₀, optical density at 600 nm.

When DPA was added to the culture as a concentrated solution in methanol, it formed a fine precipitate. It is likely that the smaller DPA particles dissolved and were transformed while the larger particles remained suspended and dissolved much more slowly. Therefore, estimates of the DPA concentration determined by HPLC, as shown in Fig. 3, must be considered approximate. Three additions of DPA totaled 1.5 mM, and about 0.15 mM DPA remained at the end of the reaction, which led to a calculated conversion efficiency of 75%. The overall rate of transformation was 4.8 μmol min⁻¹ mg of protein⁻¹. When the DPA concentrations became negligible, the concentrations of RFP declined, which indicates that RFP was removed from the solution either by decomposition or by further metabolism. The instability of RFP required that the transformation be carried out in a relatively short time. When RFP was dissolved in MSB without cells, 38% of the RFP disappeared within 2 h.

The methanol used to dissolve DPA was toxic to the culture when concentrations of DPA greater than 0.5 mM were required. When methanol was replaced by hexane as the solvent for DPA, higher concentrations of DPA could be added to the culture, and the decrease in RFP production associated with the decrease in the DPA concentration was eliminated (Fig. 3C).

Determination of the pathway for toluene degradation by Acinetobacter sp. strain F4.

Cells grown overnight on toluene were harvested, washed, and tested for the ability to oxidize several intermediates of the various pathways known to be used by bacteria for degradation of toluene (15). The rapid oxidation of 3-methylcatechol and the failure of the cresols, benzyl alcohol, and benzoate to stimulate oxygen uptake provide strong evidence that the pathway used is the toluene dioxygenase pathway (10) (Table 1).

TABLE 1.

Oxygen uptake by washed cells of Acinetobacter sp. strain F4 after growth on toluene

Substrate	Oxygen uptake (nmol/min/mg of protein)a
Toluene	708-986
Benzene	44-148
Benzyl alcohol	18-118
Benzaldehyde	24-59
Benzoate	79-83
3-Methylcatechol	687-918
Catechol	128-182
o-Cresol	9-39
m-Cresol	29-67
p-Cresol	3-39

^aRanges of typical values.

Extraction and characterization of the ring fission product.

The putative RFP was extracted from culture fluids and purified. It had a melting point of 110.9 to 112°C, accompanied by decomposition, and it produced a single peak during HPLC. When the compound was dissolved in phosphate buffer, it gave a UV-visible spectrum typical of extradiol ring fission products (9), with maxima at 431, 278, and 226 nm (Fig. 4). The yellow color was eliminated by acidification. APCI-MS analysis revealed the expected parent ion at m/z 243 (M+H⁺). MS-MS of the m/z 243 ion gave major fragments at m/z 225 ([MH₂O]⁺), 197 ([M-HCOOH]⁺), and 129 {[M-(CH)₃CHOCOOH⁺]}.

FIG. 4.

UV-visible spectra of purified RFP at alkaline and acid pH values. The dashed line is the spectrum of a sample that was acidified to pH 3 and then returned to pH 8.

Conversion of RFP to 6-phenylacetylene picolinic acid.

The purified compound was dissolved in 8 N NH₄OH and kept overnight at 60°C for conversion to 6-phenylacetylene picolinic acid. Chemical conversion of 1,688 μM RFP resulted in 931 μM 6-phenylacetylene picolinic acid or a 55% yield, which is consistent with yields reported by Asano et al. (1) for conversion of 2-hydroxymuconic semialdehyde to picolinic acid under similar conditions. The 6-phenylacetylene picolinic acid was not produced under normal culture conditions, as has been reported for other picolinic acids (3, 4, 13, 29). When 6-phenylacetylene picolinic acid was purified and extracted as described in Materials and Methods, it had a melting point of 138.2 to 139.2°C, accompanied by decomposition. CHN analysis gave values of 75.13 ± 0.18, 4.25 ± 0.06, and 6.29 ± 0.11 (averages ± standard deviations) for carbon, hydrogen, and nitrogen, respectively, which are consistent with the theoretical values of 75.33, 4.06, and 6.27, respectively. The UV-visible spectrum had absorbance maxima at 300 and 277 nm (Fig. 5). Liquid chromatography-MS analysis gave the expected molecular ion at m/z 224. Liquid chromatography-MS-MS analysis gave major fragments at m/z 178 ([M-HCOOH]⁺) and at m/z 196, the product of a likely ion-molecule reaction of m/z 178 and water (m/z 178 + 18). The ¹H and ¹³C NMR spectra (Table 2) were consistent with predicted shifts for 6-phenylacetylene picolinic acid but not for the only other possible isomer, 2-(2-phenylpropynyl)nicotinic acid. Based on the elemental analysis, MS and NMR spectra, and known reactions of the toluene dioxygenase pathway, we identified the final product as 6-phenylacetylene picolinic acid.

FIG. 5.

UV-visible spectra of purified 6-phenylacetylene picolinic acid at alkaline and acid pH values. The dashed line is the spectrum of a sample that was acidified to pH 3 and then returned to pH 8.

TABLE 2.

¹³C and ¹H NMR data for 6-phenylacetylene picolinic acid

Phenylethynyl-substituted aromatic systems for preparation of thermostable resins in which the phenylethynyl groups are oriented ortho to each other have lower temperatures for the onset of polymerization, and thus the hazards of handling are reduced (26). Replacement of the phenyl group with a pyridine should enhance the reactivity of the carbon-carbon triple bond in polymerization reactions. A related compound, 2-methyl-6-(phenylethynyl)-pyridine, is a potent and selective antagonist for certain receptors implicated in neurological and psychiatric disorders (8). 6-Phenylacetylene picolinic acid or its analogs might also be useful compounds in these systems.

Toluene dioxygenase pathway and DPA transformation.

Keener et al. (18) recently proposed that production of a yellow product from phenylacetylene by bacteria grown on toluene can be considered strong evidence that there is a pathway which is initiated by toluene dioxygenase and involves subsequent participation of a dihydrodiol dehydrogenase and an extradiol ring fission dioxygenase. Our results seem to indicate that a similar conclusion can be drawn for the transformation of DPA. We did not characterize the pathways in all of the strains that produced a yellow product from DPA, but we obtained strong evidence that the toluene dioxygenase pathway operates in Acinetobacter sp. strain F4. It seems clear, however, that not all strains that contain the toluene dioxygenase pathway can transform DPA to 2-hydroxy-6-phenylacetylene muconic semialdehyde because we failed to detect transformation with P. putida F1, the strain in which toluene dioxygenase was first described (10), or with E. coli(pDTG603) after induction of the toluene dioxygenase pathway genes with isopropyl β-d-thiogalactoside. Because the DPA molecule is considerably larger than toluene or phenylacetylene, it is remarkable that it enters the cell and that all three of the enzymes involved in the transformation can catalyze the reactions with the unusual substrate. Apparently, the binding pockets of the toluene dioxygenase, the toluene dihydrodiol dehydrogenase, and the 3-methylcatechol 2,3-dioxygenase are large enough to accommodate phenyl compounds substituted with groups as large as phenylacetylene. Because the transformation requires multiple enzymes, it is not possible at this point to determine which step(s) is inhibited by toluene. It seems clear that the enzyme that acts on the 2-hydroxymuconic semialdehyde produced from toluene is not very active with the RFP from DPA.

We established that DPA can be converted to the corresponding meta ring fission product by a strain of Acinetobacter that has the toluene dioxygenase pathway (Fig. 6). The conversion efficiency is high, but the rates are modest. The possible bottlenecks that prevent high transformation rates include insolubility of the substrate, transport limitations, competition with toluene for the active site of toluene dioxygenase, instability of RFP, and low affinity of one or more of the enzymes for the intermediates produced from DPA. We are currently working to identify and overcome the bottlenecks. It remains to be seen to what extent strain F4 can be used as a catalyst for preparation of a broad range of novel substituted picolinic acids.

FIG. 6.

Proposed transformation pathway from DPA to 6-phenylacetylene picolinic acid. The toluene dioxygenase, toluene dihydrodiol dehydrogenase, and 3-methylcatechol 2,3-dioxygenase of Acinetobacter sp. strain F4 convert DPA to the ring fission product 2-hydroxy-6-phenylacetylene muconic semialdehyde. The ring fission product is chemically converted to 6-phenylacetylene picolinic acid by addition of ammonia.

Inclusion of the normal growth substrate to support the transformation is a strategy that might have applications in other systems. Conversion of 3-chloro-2,5-dimethylpyrazine to 5-methylpyrazinecarboxylic acid has been reported for cells growing on xylene (20), although details concerning the amount of xylene required for the system were not disclosed. The transformation of a nongrowth substrate by bacterial cells growing on a related growth substrate is called cooxidation. This term was originally used by Leadbetter and Foster (23) to describe oxidation of related hydrocarbons by bacteria growing on methane. The process was discussed in detail in a review by Perry (27). In the review Perry also described a considerable amount of work on the transformation of a variety of substrates by bacteria grown on hexadecane as the carbon source. When the enzymes involved in the degradation of the growth substrate are also responsible for the transformation of the nongrowth substrate, the two substrates are competitive inhibitors and the efficiency of the transformation is difficult to predict. More recently, competitive inhibition has been described for methyl tert-butyl ether transformation by cells grown with butane (24) and for trichoroethylene transformation by cells grown with a variety of primary substrates (2, 7, 25). Therefore, most transformation studies have been done with nonproliferating cells grown on the primary substrate and switched to the substrate of interest (19). Such a strategy limits the time during which the cells are active because they quickly deplete energy reserves and the relevant enzymes often do not remain induced. To maintain induction, strategies have been devised that alternate between supplying the inducer and supplying the contaminant of interest (30). Alternatively, it is common to express the necessary genes in a foreign host and conduct the transformations with an unrelated carbon source as the growth substrate and/or inducer or to derive strains that are constitutive for expression of the necessary genes (21). Our results indicate that it is possible to select conditions that minimize competitive inhibition and allow efficient transformation during cooxidation with wild-type cells, as previously demonstrated for trichloroethylene cometabolism (22). This strategy allows use of an inexpensive hydrocarbon to support the initial growth of the biomass and then to provide energy and maintain induction of the necessary enzymes during the subsequent transformation. This strategy might not be the most efficient strategy for industrial fermentations, but it has been proven to be very effective for screening and bench-scale studies. It has recently been reported that toluene inhibits the transformation of d-limonene in toluene-degrading strains (5). It remains to be seen whether lower concentrations of the primary substrate might be used to sustain other reactions while minimizing competitive inhibition.