Molecular cloning and characterization of cytochrome P450 and ferredoxin genes involved in bisphenol A degradation in Sphingomonas bisphenolicum strain AO1

Aims: To clone and characterize the genes bisdA and bisdB, encoding Ferredoxin(bisd) (Fd(bisd)) and cytochrome P450(bisd) (P450(bisd)), respectively, from the bisphenol A (BPA) degrading Sphingomonas bisphenolicum strain AO1. Methods and Results: The 3.7 kb region containing bisdA and bisdB was cloned by genome walking and colony hybridization. The deduced N-terminal amino acid sequences of bisdA and bisdB were consistent with those of Fd(bisd) and P450(bisd) proteins characterized in our previous report. Two transposase genes, tnpA1 and tnpA2, were also located upstream and downstream of bisdAB. From amino acid sequence analysis, P450(bisd) has two conserved regions corresponding to the oxygen and heme binding regions of the bacterial cytochrome P450 family. Fd(bisd) was similar to putidaredoxin-type [2Fe-2S] ferredoxins. Escherichia coli BL21 (DE3) cells bearing bisdB- and bisdAB-recombinant pET19b were able to degrade BPA. A spontaneous mutant, strain AO1L, which was unable to degrade BPA, was isolated from the stock culture, and it was confirmed that strain AO1L had no bisdAB region. Conclusions: P450(bisd) monooxygenase sytem, encoded by bisdAB, is one system required for BPA hydroxylation in S. bisphenolicum strain AO1. Significance and Impact of the Study: Our results indicate that bisdAB are key genes for BPA degradation in S. bisphenolicum strain AO1.