A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N- nor the C-terminal portions but only the core region of the large or a-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO a-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.