Engineering nitrogen self-sufficient cocultures of ammonium-secreting Azotobacter vinelandii and glucose-secreting Escherichia coli
Date
2019
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Interest in engineering nitrogen-fixing microbes, or diazotrophs, to secrete ammonium has historically been motivated by a desire to employ these microbes as a more sustainable source of nitrogen fertilizer to offset our current agricultural reliance on the Haber-Bosch process. However, reductionist studies of diazotrophs paired with non-native partners have also been of interest as simpler systems for studying obligatory mutualism in a highly controlled environment. In this work, we sought to investigate the metabolic underpinnings of diazotrophy that enable nitrogen self-sufficiency in a simple, two-species consortium. ☐ The compatibility of cross-fed substrates is paramount in the design of robust cocultures, often requiring genetic manipulation to override native preferences of each partner. While diazotrophs like Azotobacter vinelandii have successfully been engineered to secrete ammonium, these strains also exhibit a high substrate demand and overall inefficient carbon metabolism. Indeed, through a combination of physiological characterization and network modeling, we demonstrate that the majority (≥ 85%) of the carbon consumed by A. vinelandii is spent on respiration, ultimately to protect its nitrogenase from oxygen, rather than ATP production for fueling nitrogen fixation and other cellular processes. Thus, a major challenge in engineering stable cocultures with nitrogen-fixers is to identify partners that can meet the high substrate demands of these diazotrophs while receiving limited nitrogen in return. ☐ Furthermore, to be able to study metabolic interdependence, it was critical in our efforts to choose an external carbon source, such as xylose, that could be utilized by the partner but not A. vinelandii. Thus, the diazotroph would be forced to rely on the partner to “pre-digest” xylose into a useable form, such as glucose, while the partner in turn relies on A. vinelandii for fixed nitrogen in the form of ammonium. ☐ By deleting four genes in central metabolism, we rewired Escherichia coli to not only preferentially consume xylose over glucose, but also secrete 22-34% of the carbon it uptakes as xylose into glucose. Furthermore, by employing adaptive evolution, we obtained a strain than exhibited continuous glucose production even under complete nitrogen starvation. Through whole-genome sequencing and comparative genomics, we determined that this altruistic phenotype likely arose from a single-nucleotide mutation in XylR, the transcriptional activator of xylose catabolism, that ultimately promotes enhanced xylose-to-glucose conversion. Subsequent nutrient limitation studies identified additional media formulations that enhance this desirable phenotype. ☐ To track the population dynamics of our cocultures, we developed three independent methods for quantifying the population composition of the cocultures based on (1) proteinogenic amino acid composition, (2) acetyl-CoA labeling inferred from isotopomer spectral analysis (ISA), and (3) 13C-coculture metabolic flux analysis (13C-CMFA). These population studies unexpectedly revealed that E. coli initially dominates these cocultures, demonstrating that A. vinelandii shares the majority of its fixed nitrogen supply with E. coli. Furthermore, although no common carbon-containing compounds accumulated in the growth medium, 13C-CMFA allowed us to identify acetate as an additional cross-fed nutrient, which would not have been observable from conventional extracellular medium studies alone. ☐ In subsequent cocultures with our engineered glucose-secreting E. coli strains, we were able to test the limits of bi-directional cross-feeding by forcing each organism to rely on one another for either their carbon or nitrogen source. Under these conditions, we find that the key factor determining coculture performance is the ability of E. coli to secrete glucose under nitrogen starvation. Indeed, we found that the glucose productivities of E. coli measured under normal (i.e. nitrogen-replete) conditions were a poor predictor of strain performance in the cocultures. Altruistic glucose secretion by E. coli appears to be a complex trait, arising from multiple genotypes and environmental factors, of which enhanced XylR activity and nitrogen limitation only play a small part. ☐ Taken together, in this work, we demonstrate the capacity of engineered diazotrophs to fully meet the nitrogen requirements of another organism when cultivated together. Our investigation sheds light on the design features that enable obligate mutualism in diazotrophic cocultures. Our findings contribute towards larger efforts to understand the steep metabolic costs associated with microbial nitrogen fixation and how to best meet them towards the broader ambition of engineering more sustainable food systems by employing diazotrophs as “living fertilizers.”
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Keywords
Diazotrophy, Engineered cocultures, Metabolic engineering, Metabolic modeling, Mutualism, Nitrogen fixation