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 CuI 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.