Biotransformation of Various Substituted Aromatic Compounds to Chiral Dihydrodihydroxy Derivatives

The aerobic bacterial degradation of nonactivated aromatic compounds is usually initiated by dioxygenases that incorporate two hydroxyl groups into the aromatic substrate; the products of such reactions are chiral cis-dihydrodihydroxy derivatives, which also are called cis-dihydrodiols (10). The oxidation of aromatic compounds to cis-dihydrodiols is of special interest for biotechnological as well as chemical applications, because single cis-dihydrodiols have been shown to be valuable building blocks for stereoselective synthesis of biologically active molecules containing multiple chiral centers (6, 8, 12, 16, 18, 22). Several multicomponent dioxygenases that dihydroxylate aromatic compounds have been described (7). Toluene dioxygenases (TDO), naphthalene dioxygenase (NDO), and biphenyl dioxygenase (BDO) are the best known members of this class of dioxygenases (12). The components of these oxygenase complexes have been purified, and the encoding genes have been cloned and expressed in Escherichia coli (12). Experiments on substrate specificity showed that TDO from Pseudomonas putida F39/D and P. putida UV4 oxidize monosubstituted benzenes, except fluorobenzene, to 3-substituted cis-benzene dihydrodiols with an S configuration at the C-1 atom (3, 4, 15). BDO from Pseudomonas sp. strain LB400 dihydroxylates and dechlorinates chlorinated biphenyls (11) to the respective 2,3 or 3,4 cis-diols, whereby dihydroxylation and dechlorination happen on both phenyl rings. NDO from Pseudomonas sp. strain NCIB 9816 has a relaxed substrate specificity and catalyzes the dioxygenation of naphthalene to (+)-cis-(1R,2S)dihydroxy-1,2-dihydronaphthalene. Many related 2- and 3-ring aromatic and hydroaromatic compounds are also substrates and are turned over to the respective cis-diols (14). A mutant of the not-so-well-characterized Pseudomonas fluorescens N3 was used in the bioconversion of several naphthalene derivatives to the corresponding cis-dihydrodiols on a milligrams-to-grams scale (2). A recent review lists more than 140 diols that have been described to date (12). Although only a few of them have been used as synthons, the further development of the area depends on the discovery of novel dioxygenase reactions and on the development of biotechnological processes to produce the different metabolites. Werlen et al. recently described the cloning and expression in E. coli DH5α(pTCB144) of the chlorobenzene dioxygenase (CDO) of Pseudomonas sp. strain P51 (21). Analysis of the genes showed that CDO is a three-component aromatic ring dioxygenase. It consists of the gene products of tcbAa, encoding the large subunit of the terminal oxygenase; tcbAb, encoding the small subunit; tcbAc, encoding the ferredoxin; and tcbAd, encoding the NADH reductase. Homology comparisons indicate that these genes and gene products are most closely related to those of TDO of P. putida F1 (todC1C2BA) and are distantly related to those of NDO and BDO (21). Here we report the ability of the CDO system of the recombinant E. coli DH5α(pTCB144) to oxidize different classes of aromatic compounds to the corresponding cis-dihydrodiols. We characterized each of the 48 reaction products as thoroughly as possible. The CDO system turned out to be well suited for the production of dihydrodiols, and further work to scale up production is under way.