Evolving a new electron transfer pathway to nitrogenase in an alphaproteobacterium
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ABSTRACT:
PROVIDER: PRJNA858255 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR20138890_1.fastq.gz | Fastqsanger.gz | |||
| SRR20138890_2.fastq.gz | Fastqsanger.gz | |||
| SRR20138891_1.fastq.gz | Fastqsanger.gz | |||
| SRR20138891_2.fastq.gz | Fastqsanger.gz | |||
| SRR20138892_1.fastq.gz | Fastqsanger.gz |
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Project description:Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin or flavodoxin to reduce dinitrogen gas (N2) to produce ammonia and hydrogen. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes many proteins that can reduce ferredoxin, the electron bifurcating FixABCX complex is the only one shown to support nitrogen fixation. To gain insight into why R. palustris is unable to use these other enzymes to reduce ferredoxin in the absence of FixABCX, we isolated a suppressor of R. palustris DfixC that allowed this strain to grow under nitrogen-fixing conditions. We found two mutations were necessary and sufficient to restore growth under nitrogen-fixing conditions in the absence of a functional FixABCX. One mutation was in the primary ferredoxin involved in nitrogen fixation, fer1, and the other mutation was in rpa0678, a homolog of NAD+-dependent ferredoxin:NADPH oxidoreductase, which carries out flavin-based electron bifurcation to generate reduced Fd. We present evidence that Rpa0678 plays a role in electron transfer to benzoyl-CoA reductase, the key enzyme involved in anaerobic aromatic compound degradation. Together these findings indicate that the electron transfer pathway for anaerobic aromatic compound degradation was re-purposed to support nitrogen fixation in the suppressor strain.
2022-07-19 | GSE208087 | GEO
Project description:Nitrogenases are the only enzymes able to ‘fix’ gaseous nitrogen into bioavailable ammonia and, hence, are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited, with electron transport to the iron (Fe)-nitrogenase having hardly been investigated. Here, we characterised the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain (nifD), compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterised by monitoring diazotrophic growth and nitrogenase activity in vivo. Only deletion of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviours of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.
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Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.
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Project description:Engineered cell elongation promotes extracellular electron transfer of Shewanella oneidensis
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