Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

Abstract

Background: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166.

Results: Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain.

Conclusions: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Keywords: Biological nitrogen fixation; Luciferase assay; Metabolic engineering; Promoters; Pseudomonas stutzeri; RNA-seq.

Fig. 1

Luciferase assay of constitutive promoters in P. stutzeri DSM4166 in the LB medium under the aerobic condition. (A) Schematic of the plasmid containing different promoters. pBBR1: replicon; kan: kanamycin resistance gene; firefly: firefly luciferase reporter gene. (B) Luciferase activity of plasmids containing different promoters in DSM4166. Vector: the promoterless plasmid. P_genta: the promoter of the gentamicin resistance gene. P_nifA: the promoter of the nitrogen fixation pathway specific positive regulator gene nifA. Error bars indicate the standard deviations of three replicates (n = 3). The P-value cutoff for all the plots is 0.05. * P < 0.05, ** P < 0.01, and *** P < 0.001. One-way ANOVA test with Tukey Pairwise comparisons was used to compute statistical significance

Fig. 2

Luciferase activity of plasmids containing different promoters in DSM4166 in the nitrogen free medium K under the under the microaerobic (1% oxygen) condition. Vector: the promoterless plasmid. P_genta: the promoter of the gentamicin resistance gene. P_nifA: the promoter of the nitrogen fixation pathway specific positive regulator gene nifA. Error bars indicate the standard deviations of three replicates (n = 3). The P-value cutoff for all the plots is 0.05. * P < 0.05, ** P < 0.01, and *** P < 0.001. One-way ANOVA test with Tukey Pairwise comparisons was used to compute statistical significance

Fig. 3

Enhanced nitrogenase activity by overexpressing nifA using endogenous strong constitutive promoters in DSM4166. (A) The nitrogenase activity of DSM4166 and nifA overexpression strains cultivated in the nitrogen-free medium K at 1% oxygen determined by the acetylene reduction method. (B) The concentration of extracellular ammonium produced by DSM4166 and nifA overexpression strains. (C) The transcriptional level of selected 25 nif genes in the P12445-nifA overexpression strain relative to DSM4166 cultivated in the same condition as A. Error bars indicate the standard deviations of three replicates (n = 3). The P-value cutoff for all the plots is 0.05. * P < 0.05, ** P < 0.01, and *** P < 0.001. ns, not significant. One-way ANOVA test with Tukey Pairwise comparisons was used to compute statistical significance

Fig. 4

The growth curve of DSM4166 and nifA overexpression strains cultivated in the medium K at 99% N₂ and 1% O₂