Characterization of a dszEABC operon providing fast growth on dibenzothiophene and construction of broad-host-range biodesulfurization catalysts

Abstract

A new operon for biodesulfurization (BDS) of dibenzothiophene and derivatives has been isolated from a metagenomic library made from oil-contaminated soil, by selecting growth of E. coli on DBT as the sulfur source. This operon is similar to a dszEABC operon also isolated by metagenomic functional screening but exhibited substantial differences: (i) the new fosmid provides much faster growth on DBT; (ii) associated dszEABC genes can be expressed without the need of heterologous expression from the vector promoter; and (iii) monooxygenases encoded in the fosmid cannot oxidize indole to produce indigo. We show how expression of the new dszEABC operon is regulated by the sulfur source, being induced under sulfur-limiting conditions. Its transcription is activated by DszR, a type IV activator οf σN-dependent promoters. DszR is coded in a dszHR operon, whose transcription is in turn regulated by sulfur and presumably activated by the global regulator of sulfur metabolism CysB. Expression of dszH is essential for production of active DszR, although it is not involved in sulfur sensing or regulation. Two broad-host-range DBT biodesulfurization catalysts have been constructed and shown to provide DBT biodesulfurization capability to three Pseudomonas strains, displaying desirable characteristics for biocatalysts to be used in BDS processes.