Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 1802 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen.Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these eflectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.The degradation of toluene by bacteria incorporating molecular oxygen into toluene has been studied by several laboratories, resulting in the characterization of four pathways in pseudomonads. The best characterized of these pathways is the TOL plasmid pathway of Pseudomonas putida mt-2 (25). The TOL plasmid pathway converts toluene to benzyl alcohol, benzaldehyde, benzoate, and finally to catechol, which undergoes meta cleavage. The structural and regulatory genes of this pathway have been mapped and consist of two regulons. The upper pathway operon, xylCMAB, encodes enzymes for the metabolism of toluene to benzoate and is positively regulated byxylR (7,11,24) together with the sigma factor NtrA (5). The lower pathway enzymes for the metabolism of catechol are encoded by the xylXYZLTEGFJQKIH operon (9) and regulated by xylS (6,10,19,24). Transcription of xylS is also mediated by xylR and the NtrA sigma factor, which results in overproduction of xylS and subsequent transcription of the meta-cleavage pathway operon in the absence of meta-cleavage intermediates (19). The enzymes of the upper pathway also exhibit broad substrate specificity, transforming not only toluene and xylene but also ethyl-, methyl-and chloro-substituted toluene (1).P. putida Fl also metabolizes toluene; however, the first step in the pathway is the transformation of toluene to cis-toluene dihydrodiol, followed by conversion to 3-methylcatechol, which undergoes meta cleavage. The genetic organization (29, 30) and biochemistry (8,20,27) of the enzymes responsible for the catabolism of toluene to 3-methylcatechol have been studied extensively. The toluene 2,3-dioxygenase of P. putida Fl metabolizes a wide range of hydrocarbon...

Microbial biocatalysis is used in the commercial production of many flavor and fragrance chemicals. Bulk flavoring chemicals such as citric acid, high fructose corn syrup, and glutamic acid are produced in millions of pounds annually using microbial processes. In the past few years, biocatalysis has also begun to play an increasingly important role in the production of many flavor and fragrance aroma chemicals. Microbial processes have traditionally played an integral role in the development of complex mixtures of flavor and aroma chemicals since the discovery of beer, wine, cheese, and soy sauce thousands of years ago. Today, contemporary microbiological techniques are being increasingly applied to enhance the efficiency of many microbial biocatalysts for the production of specific flavor and fragrance chemicals. However, to ensure commercial implementation of these new microbial processes, much more needs to be learned about the basic biochemistry and genetics of these novel biocatalysts.

The closely linked structural genes (fdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, fdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PA04032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.

Plasmid pRO101, a derivative of plasmid pJP4 which contains Tnl721 inserted into a nonessential region, is inducible for 2,4-dichlorophenol hydroxylase (DCPH) encoded by tfdB. Plasmid pRO103, which has a deletion in the BamHI-F-BamHI-E region of plasmid pRO101, has elevated basal levels of DCPH but is uninducible. The regulatory gene for tfdB, designated tfdS, was cloned as an 8.3-kilobase-pair EcoRI-E fragment. When the cloned tfdS gene was in trans with plasmid pRO103, the baseline DCPH levels were repressed to normal uninduced levels and were fully induced when this strain was grown in the presence of 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol, or 4-chlorocatechol. However, when tfdS was in trans with tfdB in the absence of tfdCDEF, tfdB was repressed but could not be induced. When tfdS and tfdCl, which encodes chlorocatechol 1,2-dioxygenase, are in trans with tfdB, tfdB remained uninduced, indicating that a downstream metabolite of chloro-cis,cis-muconate, either 2-cis-chlorodiene lactone or chloromaleylacetic acid, is the effector. Collectively, these data demonstrate that the gene product of tfdS acts as a repressor of tfdB in the absence of an effector and as an activator of tfdB when an effector is present.The physiology, biochemistry, and genetics of 2,4-dichlorophenoxyacetic acid (TFD) degradation have been studied by many laboratories. Initial studies have focused on the delineation of the pathway for TFD degradation (8,9,25,28). These studies have shown that TFD is first converted to 2,4-dichlorophenol (DCP) and subsequently hydroxylated to chlorocatechol. Chlorocatechol is then metabolized through a modified ortho-cleavage pathway, resulting in the production of chloromaleylacetic acid (Fig.

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