NosZ gene cloning, reduction performance and structure of Pseudomonas citronellolis WXP-4 nitrous oxide reductase

Abstract

Nitrous oxide reductase (N₂OR) is the only known enzyme that can reduce the powerful greenhouse gas nitrous oxide (N₂O) to harmless nitrogen at the final step of bacterial denitrification. To alleviate the N₂O emission, emerging approaches aim at microbiome biotechnology. In this study, the genome sequence of facultative anaerobic bacteria Pseudomonas citronellolis WXP-4, which efficiently degrades N₂O, was obtained by de novo sequencing for the first time, and then, four key reductase structure coding genes related to complete denitrification were identified. The single structural encoding gene nosZ with a length of 1914 bp from strain WXP-4 was cloned in Escherichia coli BL21(DE3), and the N₂OR protein (76 kDa) was relatively highly efficiently expressed under the optimal inducing conditions of 1.0 mM IPTG, 5 h, and 30 °C. Denitrification experiment results confirmed that recombinant E. coli had strong denitrification ability and reduced 10 mg L⁻¹ of N₂O to N₂ within 15 h under the optimal conditions of pH 7.0 and 40 °C, its corresponding N₂O reduction rate was almost 2.3 times that of Alcaligenes denitrificans strain TB, but only 80% of that of wild strain WXP-4, meaning that nos gene cluster auxiliary gene deletion decreased the activity of N₂OR. The 3D structure of N₂OR predicted on the basis of sequence homology found that electron transfer center CuA had only five amino acid ligands, and the S2 of the catalytically active center CuZ only bound one Cu_I atom. The unique 3D structure was different from previous reports and may be closely related to the strong N₂O reduction ability of strain WXP-4 and recombinant E. coli. The findings show a potential application of recombinant E. coli in alleviating the greenhouse effect and provide a new perspective for researching the relationship between structure and function of N₂OR.