Genetic and biochemical characterization of two new extradiol dioxygenases of Sphingomonas sp. strain RW1 and construction of two gene cassettes for biodegradation of dibenzofuran and dioxin

The major goal of this study was the development of two gene cassettes carrying all genes for dibenzo-p-dioxin and dibenzofuran degradation upper pathway from Sphingomonas sp. strain RW1. Such cassettes should be used to create organisms capable to entirely mineralise chlorodioxins and chorodibenzofurans. To this end, the dioxin and dibenzofuran catabolism in RW1 strain was initially investigated on a genetic and biochemical level. It could be shown that at least three different extradiol dioxygenases are encoded in the RW1 genome, namely DbfB, Edo2 and Edo3. All of them were rapidly inactivated during transformation of the substrate 2,2´,3-trihydroxybiphenyl ether (THBE) into catechol and 2-pyrone-6-carboxylate, identified as reaction products. The inactivation was dependent on the presence of oxygen and substrate turnover and was very effective for all enzymes, but less pronounced for Edo3, and especially Edo2. Based on the kinetic studies, the appropriate extradiol dioxygenases coding genes to be included in the gene cassettes have been selected. The dibenzofuran gene cassette, comprising dbfB, was performing properly as evidenced by quantitative transformation of dibenzofuran into salicylate. Both gene cassettes transformed dibenzo-p-dioxin into THBE only, due to inactivation of the extradiol dioxygenases. By increasing the concentration of Edo2 in the transformation test it could be verified that Edo2 is suited, however imperfectly, for the further transformation of THBE.