Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity.
Biological nitrogen fixation
Luciferase assay
Metabolic engineering
Promoters
Pseudomonas stutzeri
RNA-seq
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
03 May 2023
03 May 2023
Historique:
received:
08
12
2022
accepted:
09
04
2023
medline:
5
5
2023
pubmed:
4
5
2023
entrez:
3
5
2023
Statut:
epublish
Résumé
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. 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. 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.
Sections du résumé
BACKGROUND
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
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
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.
Identifiants
pubmed: 37138314
doi: 10.1186/s12934-023-02085-3
pii: 10.1186/s12934-023-02085-3
pmc: PMC10155442
doi:
Substances chimiques
Nitrogen
N762921K75
Nitrogenase
EC 1.18.6.1
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
91Subventions
Organisme : National Key Research and Development Program of China
ID : 2021YFC2101000
Organisme : the 111 Project
ID : B16030
Organisme : National Natural Science Foundation of China
ID : 32122049
Informations de copyright
© 2023. The Author(s).
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