Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45°C and 55°C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.