Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae

Ambrosio, Rafael; Ortiz‐Marquez, Juan C.; Curatti, Leonardo

doi:10.1016/j.ymben.2017.01.002

Cited by 64 publications

(43 citation statements)

References 39 publications

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“…We investigated potential competing pathways that we hypothesized would become exacerbated under the conditions associated with the high ammonium accumulating phenotype, and might result in energetically costly and futile cycles that inadvertently limit BNF, such as the ammonium importer, AmtB [21,22]. A description of these parameters and pathways that were targeted, and the findings associated with them, are presented herein, and demonstrate a potential to further improve ammonium production by the strain, and yield additional products that could further enhance the economic potential of pursuing routes to biofertilizer production through BNF [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

2020

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Background: The obligate aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. Results: In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. Conclusions: These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

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Section: Introductionmentioning

confidence: 99%

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

2020

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“…This behaviour can be due to most diazotrophs that use concerted mechanisms for cellular N homeostasis and typically do not fix N in excess nor excrete significant amounts of N-fixation products. The factors required for nitrogen fixation are to date quite defined and therefore the use of genetically modified diazotrophs to improve plant growth has been proposed (Van Dommelen et al, 2009;Geddes et al, 2015;Ambrosio et al, 2017). Several studies have also highlighted that the beneficial effects of microbes on plants growth can be enhanced by coinoculation with different microorganisms.…”

Section: Introductionmentioning

confidence: 99%

Efficient colonization of the endophytes Herbaspirillum huttiense RCA24 and Enterobacter cloacae RCA25 influences the physiological parameters of Oryza sativa L. cv. Baldo rice

Andreozzi

Prieto

Mercado‐Blanco

et al. 2019

Environmental Microbiology

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Summary Several important bacterial characteristics, such as biological nitrogen fixation, phosphate solubilization, 1‐aminocyclopropane‐1‐carboxylate deaminase activity and production of siderophores and phytohormones, can be assessed as plant growth promotion traits. Our aim was to evaluate the effects of nitrogen fixing and indole‐3‐acetic acid (IAA) producing endophytes in two Oryza sativa cultivars (Baldo and Vialone Nano). Three bacteria, Herbaspirillum huttiense RCA24, Enterobacter asburiae RCA23 and Staphylococcus sp. 377, producing different IAA levels, were tested for their ability to enhance nifH gene expression and nitrogenase activity in Enterobacter cloacae RCA25. Results showed that H. huttiense RCA24 performed best. Improvement in nitrogen fixation and changes in physiological parameters such as chlorophyll, nitrogen content and shoot dry weight were observed for plants co‐inoculated with strains RCA25 and RCA24 in a 10:1 ratio. Based on confocal laser scanning microscopy analysis, strain RCA24 was the best colonizer of the root interior and the only IAA producer located in the same root niche occupied by RCA25 cells. This work shows that the choice of a bio‐inoculum having the right composition is one of the key aspects to be considered for the inoculation of a specific host plant cultivar with microbial consortia.

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“…This strain was based on a disruption of the antiactivator nifL gene while the nifA gene and ribosome binding sites (RBSs) remained intact. Strains producing elevated levels of ammonium are of interest in many agricultural settings (6,(11)(12)(13)(14)(15)(16). We have reconstructed a similar strain by using approaches taken by Bali and coworkers (Kennedy laboratory) as a guide for producing a strain with a similar phenotype, A. vinelandii strain AZBB163 (10,12).…”

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confidence: 99%

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Barney

Plunkett

Velmurugan

et al. 2017

Appl Environ Microbiol

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Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene result in the deregulation of the transcriptional activator NifA in the nitrogen-fixing bacterium , triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

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Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae

Cited by 64 publications

References 39 publications

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

Efficient colonization of the endophytes Herbaspirillum huttiense RCA24 and Enterobacter cloacae RCA25 influences the physiological parameters of Oryza sativa L. cv. Baldo rice

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

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