Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacetrium 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-four candidate promoters were identified from DSM4166 by RNA-seq analysis. These 24 promoters were cloned and characterized using the firefly luciferase gene. The strengths of fourteen 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 23 folds. When the nifHDK nitrogenase genes and nifA were both overexpressed by endogenous strong constitutive promoters, the nitrogen fixation efficiency of DSM4166 was increased by 51 folds. 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.

Pangenomic landscapes shape the genetic circuit performance in aStutzerimonasbiodesign toolkit

“…Fifteen Stutzerimonas hosts were screened for plasmid transformability and inverter operability, in which ten were successfully transformed via electroporation with a pBBR1-KanR backbone vector (BB23). Six of these ten were chosen for further study, which are as follows: Stutzerimonas chloritidismutans NCTC10475 ( S. chloritidismutans ) 25 , Stutzerimonas perfectomarina CCUG 44592 ( S. perfectomarina ) 18 , Stutzerimonas degradans FDAARGOS 876 ( S. degradans ) 18 , Stutzerimonas stutzeri 24a13 ( S. stutzeri 24a13) 26 , Stutzerimonas stutzeri 24a75 ( S. stutzeri 24a75) 26 and Stutzerimonas stutzeri DSM 4166 ( S. stutzeri DSM 4166) 27,28 .…”

“…Fifteen Stutzerimonas hosts were screened for plasmid transformability and inverter operability, in which ten were successfully transformed via electroporation with a pBBR1-KanR backbone vector (BB23). Six of these ten were chosen for further study, which are as follows: Stutzerimonas chloritidismutans NCTC10475 (S. chloritidismutans) 25 , Stutzerimonas perfectomarina CCUG 44592 (S. perfectomarina) 18 , Stutzerimonas degradans FDAARGOS 876 (S. degradans) 18 , Stutzerimonas stutzeri 24a13 (S. stutzeri 24a13) 26 , Stutzerimonas stutzeri 24a75 (S. stutzeri 24a75) 26 and Stutzerimonas stutzeri DSM 4166 (S. stutzeri DSM 4166) 27,28 Comparative pangenomics of the selected Stutzerimonas hosts revealed almost equal sizes between the core and accessory genomes, propitiously setting the stage for investigating whether the measurable chassis-effect can be attributed to differential expression from the core or accessory genomes and which specific functions covary with host-specific device performance. Pangenome analysis of Stutzerimonas host genomes was performed using Anvi'o 29 , leading to a total of 25,344 gene calls functionally annotated using the 2020 clusters of orthologous genes (COGs) database (Fig.…”