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

Abstract

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.

Metabolic model of nitrogen-fixing obligate aerobe Azotobacter vinelandii demonstrates adaptation to oxygen concentration and metal availability

“…This model provides a framework for understanding the metabolism of A. vinelandii and is a valuable model for understanding the production of biopolymers. The model iDT1278 lacked essential enzymes required for nitrogen fixation and failed to determine an accurate growth rate in standard laboratory conditions of complete aeration and at least 10g/L of sucrose or equivalent carbon (36). A new model ( iAA1300 ) presented here builds off the model iDT1278 by adding missing reactions and manually curating inadequately annotated constraints (Table S1).…”

“…Unlocking A. vinelandii’s nitrogen fixation regulatory system by deletion of nifL gene allows nitrogenase to be constitutively expressed even in the presence of high ammonia concentration in the media (36, 49). The ability to engineer an ammonia-excreting strain has been a target for genetic engineering for many decades.…”

“…The increase of ammonia excretion essentially doubles the flux through nitrogenase and reduces the growth rate, respectively (Table 4). Ammonia-excreting strains start to excrete ammonia within the stationary phase during batch growths (36). In these conditions, cell growth would be minimal, and O 2 would be limited due to cell density.…”

“…Ammonia yields for high and low O 2 are similar with 1.3 (mol sucrose /mol ammonia ) predicted and while ∼1.4 (mol sucrose /mol ammonia ) was experimentally determined. When O 2 was increased to reduce the amount of time required for ammonia accumulation, an ammonia yield of ∼2.3 (mol sucrose /mol ammonia ) was experimentally determined, and 3 (mol sucrose /mol ammonia ) was predicted (36).…”

“…By simulating A. vinelandii to produce the maximum ammonia in agricultural or industrial scenarios, key insights can be developed for future engineering targets. To test the viability of ammonia excretion of the model and the effect of O2 maintenance, models of low and high O2 (12 and 108 µM of O2) were set to excrete ammonia at a rate of 3 mmolAmmonia • hr -1 • g CDW -1 as estimated from Plunkett et al (36). The increase of ammonia excretion essentially doubles the flux through nitrogenase and reduces the growth rate, respectively (Table 4).…”