Metabolic engineering of ammonium release for nitrogen-fixing multispecies microbial cell-factories

Ortiz‐Marquez, Juan C.; Nascimento, Mauro Do; Curatti, Leonardo

doi:10.1016/j.ymben.2014.03.002

Cited by 50 publications

(35 citation statements)

References 45 publications

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“…5B), similar to the initial reports by Bali et al (43). A more recent report by Ortiz-Marquez et al (46) coupled their A. vinelandii ⌬nifL strain previously described (9) with point mutations to glutamine synthase to yield ammonium levels up to 7 mM (46), although the long-term stability of their strain may be problematic based upon the potential for recombination events within the cell. The A. vinelandii AZBB158 and AZBB163 are also potentially problematic, since they are prone to contamination by spontaneous cheaters (cells of A. vinelandii that evolve to quit fixing nitrogen and then prey upon the ammonium released by the remaining ammonium excreting cells).…”

Section: Discussionsupporting

confidence: 74%

“…These disruptions that were shown to increase extracellular nitrogen levels when sufficient sugar is provided to the media by deleting/replacing ureABC and/or amtB could also be constructed as gene deletions using markerless techniques developed in our laboratory (to be published) or through congression approaches (23), resulting in true gene deletions that would not be transgenic but simply gene deficient. Each of these modifications could be coupled to one another or combined with approaches that alter the nifLA operon to enhance total nitrogen output further, similar to the approach taken by Ortiz-Marquez et al with nifLA alterations coupled to glnA mutations (46). The development of stable genetic constructs that lack foreign genes would yield biofertilizer strains with a greater potential for utilization in algal culture and that could be applied to current conventional agricultural crops as an enhanced biofertilizer as well.…”

Section: Discussionmentioning

confidence: 99%

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Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Barney

Eberhart

Ohlert

et al. 2015

Appl Environ Microbiol

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Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a freeliving bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. N utrient requirements are directly linked to biomass production, and any potential increased improvement in the scale of biomass yield will necessitate a proportional increase in the demand for essential nutrients. For all photosynthetic organisms (photoautotrophs such as land plants, algae, and cyanobacteria) with requisite light energy and water, nitrogen is the most limiting and expensive nutrient input for aquaculture and agricultural production alike (1). A majority of our current nitrogen fertilizer production is tied to the burning of fossil fuels to generate ammonia from molecular nitrogen (N 2 gas) through the Haber-Bosch process, which accounts for 3 to 5% of world natural gas consumption, or about 1 to 2% of total worldwide energy expenditures (1-3). In developed countries, industrial nitrogen production is accompanied by a huge economic and energetic cost overall (2), while this key nutrient limits agricultural productivity in developing countries, where energy and infrastructure costs prohibit the utilization of the Haber-Bosch process to produce ammonia from atmospheric nitrogen on a large scale.The development of biological approaches to improve biof...

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Barney

Eberhart

Ohlert

et al. 2015

Appl Environ Microbiol

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“…If the functionality of this NH 3 assimilation pathway is disrupted, direct production of extracellular NH 3 should occur. Noting that GS inhibitors can induce NH 3 secretion in sugar-fermenting diazotrophs (14), we turned to the specific GS inhibitor phosphinothricin (PPT) (42) to block the NH 3 assimilation pathway and allow NH 3 to passively diffuse out into the extracellular medium (pathway 2 in Figs. 1 and 4A).…”

Section: Resultsmentioning

confidence: 99%

“…A distributed approach toward NH 3 synthesis from renewable energy sources at ambient conditions would enable on-site deployment and reduce CO 2 emissions. To this end, significant effort has been devoted to promoting the reduction of nitrogen to NH 3 with the use of transition metal catalysts (3)(4)(5), electrocatalysts (6), photocatalysts (7-11), purified nitrogenases (N 2 ases) (11,12), and heterotrophic diazotrophs (13,14), potentially powered by renewable energy and operating at ambient conditions. Such approaches, however, typically use sacrificial reductants to drive conversion at low turnover or suffer poor selectivity.…”

mentioning

confidence: 99%

Ambient nitrogen reduction cycle using a hybrid inorganic–biological system

Liu

Sakimoto

Colón

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

194

220

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We demonstrate the synthesis of NH 3 from N 2 and H 2 O at ambient conditions in a single reactor by coupling hydrogen generation from catalytic water splitting to a H 2 -oxidizing bacterium Xanthobacter autotrophicus, which performs N 2 and CO 2 reduction to solid biomass. Living cells of X. autotrophicus may be directly applied as a biofertilizer to improve growth of radishes, a model crop plant, by up to ∼1,440% in terms of storage root mass. The NH 3 generated from nitrogenase (N 2 ase) in X. autotrophicus can be diverted from biomass formation to an extracellular ammonia production with the addition of a glutamate synthetase inhibitor. The N 2 reduction reaction proceeds at a low driving force with a turnover number of 9 × 10 9 cell -1 and turnover frequency of 1.9 × 10 4 s -1 ·cell -1 without the use of sacrificial chemical reagents or carbon feedstocks other than CO 2 . This approach can be powered by renewable electricity, enabling the sustainable and selective production of ammonia and biofertilizers in a distributed manner.nitrogen fixation | Xanthobacter | ammonia synthesis | fertilizer | solar

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“…The GS inhibitor methionine sulfoximine (MSX) provides a useful probe to analyze the influence of GS activity on growth and metabolism (22,23). The effect of this GS inhibitor has been investigated on growth and intracellular metabolite contents for both strains under nondiazotrophic conditions.…”

Section: Resultsmentioning

confidence: 99%

Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion

Mus

Tseng

Dixon

et al. 2017

Appl Environ Microbiol

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Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance.IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.KEYWORDS Azotobacter vinelandii, glnE, ammonium assimilation, nitrogen fixation, regulation, glutamine synthetase T he most common pathways of ammonium assimilation in bacteria are known to be mediated by two major pathways (1, 2): the glutamate dehydrogenase (GDH) pathway when the extracellular concentration of ammonium is high, and the glutamine synthetase (GS) and glutamate synthase (GOGAT) pathway at low ammonium concentrations. In addition, the alanine dehydrogenase (ADH) enzyme has also been suggested to be involved in ammonium assimilation in most methylotrophs (3). The GDH

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Metabolic engineering of ammonium release for nitrogen-fixing multispecies microbial cell-factories

Cited by 50 publications

References 45 publications

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Ambient nitrogen reduction cycle using a hybrid inorganic–biological system

Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion

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