Nitrogen is an essential part of many of the cell’s building blocks, including amino acids and nucleotides, which form proteins and DNA respectively. Therefore, nitrogen has to be available to cells so that they can survive and grow. In nature, some microorganisms convert the gaseous form of nitrogen into ammonium, which then acts as the nitrogen source of most organisms, including bacteria, plants and animals. Once cells take up ammonium, it is ‘fixed’ by becoming part of an amino acid called glutamate, which has a so-called ‘amine group’ that contains a nitrogen. Glutamate then becomes the central source for passing these amines on to other molecules, distributing nitrogen throughout the cell.

This coupling between ammonium fixation and glutamate production evolved over millions of years and occurs in all organisms. However, the complete metabolic network that underlies the distribution of amines remains poorly understood despite decades of research. Furthermore, it is not clear whether ammonium can be fixed in a way that is independent of glutamate.

To answer these questions, Schulz-Mirbach et al. used genetic engineering to create a strain of the bacterium E. coli that was unable to make glutamate. These mutant cells could only grow in the presence of certain amino acids, which acted as alternative amine sources. Schulz-Mirbach et al. found that enzymes called transaminases, and one called AspC in particular, were required for the cells to be able to produce glutamate using the amine groups from other amino acids. Notably, Schulz-Mirbach et al. showed that AspC, which had previously been shown to use an amino acid called aspartate as a source of amine groups, is indispensable if the cell is to use the amine groups from other amino acids – including histidine, tyrosine, phenylalanine, tryptophan, methionine, isoleucine and leucine.

Schulz-Mirbach et al. also discovered that if they engineered the E. coli cells to produce transaminases from other species, the repertoire of molecules that the cells could use as the source of amines to generate glutamate increased. In a final set of experiments, Schulz-Mirbach et al. were able to engineer the cells to fix ammonium by producing aspartate and leucine, thus entirely bypassing the deleted routes of glutamate synthesis. These data suggest that fixing ammonium and distributing nitrogen in E. coli can be very flexible.

The results from these experiments may shed light on how cells adapt when there is not a lot of ammonium available. Moreover, this study could advance efforts at metabolic engineering, for example, to create molecules through new pathways or to boost the production of amino acids needed for industrial purposes.

Nitrogen is essential for all forms of life as it is part of 75% of the cell’s building blocks (mainly in proteins and nucleic acids) (Milo and Phillips, 2015). The conversion of atmospheric dinitrogen (N₂) to ammonia (NH₃) by diazotrophic bacteria or industrially by the Haber–Bosch process is essential to make it available for assimilation by plants and other organisms to produce nitrogenous compounds.

While carbon fixation has evolved several times, resulting in versatile naturally occurring ways of carbon fixation (Löwe and Kremling, 2021), the introduction of ammonium (NH₄⁺, protonated form of ammonia) into the building blocks of life is similar in all organisms and limited to the fixation of ammonium at the node between 2-ketoglutarate, glutamate, and glutamine (Figure 1). Here, three cooperating canonical enzymes assimilate ammonium in two distinct ways. In the first reaction, glutamate is the direct product of glutamate dehydrogenase (gdhA, GDH). In the second pathway, the combined activity of glutamine synthetase (glnA, GS) and glutamate synthase (glutamine 2-ketoglutarate aminotransferase, gltBD, GOGAT) fixes another ammonium and converts glutamate into glutamine, which then donates one amine to 2-ketoglutarate to form two glutamate molecules (Helling, 1994; Kumar and Shimizu, 2010; Figure 1). To make all essential amino acids and other aminated compounds, glutamate and glutamine then donate their amines to specific keto acids or other amino acid precursors in reversible transferase reactions. As ammonium enters metabolism solely via these two routes, all cellular nitrogen is provided by either glutamate (75%) or glutamine (25%) (Yang et al., 2018).

Figure 1

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Besides the glutamate biosynthesis node, alternative entry points for ammonium theoretically exist, for example, alanine dehydrogenase or aspartate ammonia lyase, but these are not relevant for ammonium assimilation (Kim and Hollocher, 1982). Evolution has developed a system for ammonium assimilation, which is controlled by its intracellular availability. The ATP investment driving GS activity makes amination reactions favorable even at low ammonium concentrations. Additionally, most of the GS orthologs have evolved kinetic parameters optimized for lower ammonium concentrations with an apparent K_M of 0.1 mM for ammonium (Reitzer, 2014). At high ammonium concentrations, the NADPH-dependent (and thus energetically cheaper) GDH allows more efficient ammonium assimilation than GS (Reitzer, 2014). This metabolic switch might explain the prominence of glutamate-based ammonium assimilation as opposed to other ammonia entry points in nature. To generate the NADPH required for GDH, Escherichia coli mostly uses the membrane-bound proton-translocating transhydrogenase (PntAB) (Sauer et al., 2004). This enzyme exploits the proton motif force to drive proton translocation from NADH to NADP⁺ (Spaans et al., 2015), and thereby indirectly competes with ATP synthesis. Therefore, when growing under high ammonium concentrations, growth of this microorganism might benefit from ammonium assimilation via NADH-dependent dehydrogenases.

Following these thoughts, we tried to assess whether alternative routes for ammonium assimilation can arise from the metabolic network of E. coli. For this purpose, we systematically investigated the flexibility of the amination network in a glutamate auxotrophic – and hence ammonium assimilation deficient – E. coli strain. This study provides fundamental knowledge on the plasticity of ammonium metabolism in E. coli and moreover addresses industrial interests by providing a versatile bacterial chassis for screening and optimization of ammonium assimilation and transamination reactions.

Our study provides a broad investigation of amino acid and ammonium metabolism in E. coli. To examine which amino acids can be used as an amine source to compensate for the absence of canonical glutamate production, we created a glutamate auxotrophic (glut-aux) strain and tested its ability to grow on different amino acids (Figure 2, supportive summary in Figure 2—figure supplement 1). Our experiments revealed that the glut-aux did not grow on all tested amino acids. Here, we note that the substrate spectrum of the underlying amination network might be larger than identified by our experiments since for some amino acids we cannot rule out that their uptake limits their intracellular availability to the network. Specifically, this might be the case for valine, threonine, and β-alanine, which did not support growth of any strain. However, for some of these, previous studies identified the mechanisms preventing utilization as an amine source. For example, use of threonine as nitrogen source would require induction of threonine dehydrogenase by the presence of leucine (Potter et al., 1977). Growth in the presence of valine was shown to be inhibited due to valine-dependent inhibition of acetohydroxy acid synthase essential to isoleucine biosynthesis (Tuite et al., 2005). In contrast, other amino acids known to be suitable nitrogen sources for the wildtype and canonical transamination products of glutamate did not rescue growth of the glut-aux strain via the reverse reaction (i.e., alanine). For this amino acid, we identified alanine degradation as the counteracting pathway. The glut-aux strain was capable of growth on most amino acids (proline, aspartate, asparagine, methionine, leucine, isoleucine, valine, glutamate, glutamine, ornithine, tyrosine, phenylalanine, tryptophan, and histidine). As absence of nitrogen from the medium only affected growth of the glut-aux on ornithine, we concluded that nitrogen response-dependent regulation was of minor relevance for amino acid metabolism. We assumed that in order for the glut-aux strain to grow, amino acids could either (i) fully replace glutamate as an amine donor, (ii) donate their amine directly to 2-ketoglutarate, (iii) be converted into an intermediate donating an amine to 2-ketoglutarate to form glutamate, or (iv) be direct glutamate precursors. Given the inability of the selection strain to synthesize glutamate, intracellular glutamate levels are expected to be lower than in a wildtype strain under saturating nitrogen availability. Therefore, some of the identified rescue mechanisms might not be the universally favored metabolic options. However, defining the mechanisms of growth restoration in the glut-aux strain might give insights into strategies of adaptation to limiting ammonium availability. To investigate which reactions allowed amine utilization from amino acids, we deleted several genes responsible for the respective amino acid degradation pathway. While proline was directly converted to glutamate (rescue mechanism IV, Figure 1), utilization of histidine, tyrosine, phenylalanine, tryptophan, methionine, isoleucine, and leucine depended on transamination mediated by AspC, one of the three main transaminases in E. coli (Gelfand and Steinberg, 1977; rescue mechanism II, Figure 1). Three aspects of this finding were surprising to us. First, deletion of AspC alone was sufficient to abolish growth of the glut-aux strain on tyrosine, phenylalanine, isoleucine, and leucine. This was unexpected since the other two main transaminases TyrB and IlvE share cross-reactivities with these amino acids and indicates that in spite of their cross-reactivity they cannot fully replace AspC in vivo. Residual tyrosine and phenylalanine aminotransferase activity in tyrB and ilvE knockout strains in previous works (Gelfand and Steinberg, 1977) led to the hypothesis that AspC was mainly exhibiting these activities, which herewith is further supported. Second, deletion of AspC abolished growth on histidine, methionine, isoleucine, and leucine for which only low or no specific activity of AspC was reported in vitro (Hayashi et al., 1993; Powell and Morrison, 1978). Under physiological conditions, the substrate range of AspC thus seems to be broader than previously anticipated. Third, the glut-aux ΔaspC strain was unable to grow with tryptophan as an amine source. Although activity of ΑspC with tryptophan was demonstrated in vitro (Hayashi et al., 1993; Powell and Morrison, 1978), transamination is not a known tryptophan degradation pathway in E. coli (Reitzer, 2014). We hence conclude that in the glut-aux strain, tryptophan is the direct substrate of AspC to form (indole-3-yl)-pyruvate and glutamate as in some bacteria (e.g., P. aeruginosa [Bortolotti et al., 2016], Clostridium sporogenes [O’Neil and DeMoss, 1968]), protozoa (Trichomonas vaginalis [Lowe and Rowe, 1985]), or mammals (Shrawder and Martinez-Carrion, 1972). Grouping amino acids by expected rescue mechanism and rescue phenotype (Figure 2—figure supplement 1) revealed that the majority of amino acids we suggest to be used via direct transamination rescued the glut-aux strain, but were not a good amine source for the wildtype (Figure 2, Figure 2—figure supplement 1). Additionally, we found AspC to be essential for the growth of the glut-aux on all amino acids not supporting WT growth (Figure 5). Therefore, we suggest promiscuous transaminase activity here rescuing growth of the glut-aux. strain to be one mechanism for adaptation to lacking glutamate availability. Hence, our study expands previous knowledge by several novel transamination routes in E. coli, which need to be considered in contexts of low amine or glutamate availability.

In addition to identifying natively present routes of amino acid metabolism, we demonstrated how amino acid metabolism can be rewired to allow utilization of new amine sources. Overexpression of alanine-2-ketoglutarate aminotransferase, together with the removal of an alanine sink, allowed usage of alanine as an amine source. By overexpressing transaminases that transfer amines from β-alanine to 2-ketoglutarate, we engineered usage of the non-proteinogenic amino acid β-alanine as amine source. Following the same logic, we expressed an aspartate-glyoxylate aminotransferase to select for its reverse activity with glycine or serine. Upon deletion of the d-amino acid dehydrogenase gene dadA as potential internal sink for glycine and serine, the engineered strain was able to use the amine from glycine to support growth. Surprisingly, we discovered that the strain did not use glycine directly, but converted it to serine, which was yet another substrate of the transaminase showing high activity for serine (k_cat of 25.87 s^–1) with oxaloacetate as amine acceptor. In principle, the newly discovered serine-oxaloacetate transaminase activity can be coupled to formate assimilation via the reductive glycine pathway (Kim et al., 2020), which fixes ammonium by reverse activity of the glycine cleavage system. This allows ammonium transfer to make aspartate and convert serine to hydroxypyruvate instead of pyruvate (generated via serine deaminase activity in the reductive glycine pathway), hence saving ATP needed to convert pyruvate into PEP, essential for anaplerosis and gluconeogenesis. The usage of alanine, β-alanine, glycine, and serine as amine donors was engineered in the glut-aux strain by transaminase overexpression and (if necessary) removal of intracellular substrate sinks. Thus, we conclude that for these amino acids, transaminase availability was the main bottleneck rather than transport or regulatory issues. In addition to this finding, these examples demonstrate the glut-aux strain as an excellent selection platform to screen for any reaction that produces amino acids which can rescue growth. This potentially allows the discovery of new amine sources.

The function of glutamate as a universal amine transfer molecule originates in its role as canonical entry point of ammonium into metabolism (Kumada et al., 1993). We were interested in finding out whether the prominence of this ammonium assimilation mechanism can be explained by a significant advantage compared to other mechanisms of ammonium assimilation. To investigate this, we replaced canonical ammonium assimilation via glutamate biosynthesis with two alternative pathways. Both fumarate amination to aspartate and MOP amination to leucine reconstituted the ability of the glut-aux strain to assimilate free ammonium. Growth dependent on ammonium assimilation via AspA was fastest when a carbon source metabolically close to fumarate was provided. We note that assimilation of ammonium via aspartate ammonia lyase is energetically different from ammonium assimilation via glutamate. While a glutamate-dependent amination network requires NADPH consumption by glutamate dehydrogenase, an aspartate ammonia lyase-dependent network requires NADH for the reduction of oxaloacetate back to fumarate; oxaloacetate being the transamination product of aspC. NADH is energetically cheaper compared to NADPH as the recovery of the latter from NADH wastes some proton motive force through the membrane-bound transhydrogenase, and hence comes with the indirect cost of ATP synthesis (Spaans et al., 2015). Although ammonium assimilation via aspartate or leucine biosynthesis led to immediate growth of the glut-aux strain in growth-optimized laboratory conditions, these do not reflect natural conditions under which glutamate-based ammonium assimilation might have evolved and become prominent. Under ammonium-limiting conditions, GDH and GS are kinetically superior with a K_M of 2 mM and 0.1 mM for ammonia, respectively (Reitzer, 2014), compared to AspA or LeuDH from B. cereus with K_M values of 20 mM and 13 mM for ammonia, respectively (Suzuki et al., 1973; Sanwal and Zink, 1961). Additionally, the availability of two different systems (GDH and GS/GOGAT), which are each optimal for different growth conditions but generate the same molecule, is unique to glutamate-based ammonium assimilation. Ammonium assimilation as metabolic link between energy, carbon, and nitrogen metabolism requires a tight multilayer regulation of GDH and GS. Thus, immediate growth via alternative ammonium assimilation routes that are independent of native transcriptional regulation, as achieved via aspartate and leucine in our study, is nontrivial. On the other hand, the laboratory cultivation conditions that allow this immediate growth do not require the metabolic flexibility and control given by the interplay and regulation of GDH and GS. It is even likely that growth of the engineered strains under nonoptimal conditions would be impaired by a lack of flexible metabolic responses to varying nitrogen and energy availability. This might explain the conservation of glutamate-coupled ammonium assimilation as opposed to alternative ammonium assimilation pathways. Furthermore, regulatory effects, for example, GS inhibition by several amino acids (e.g., tryptophan, histidine, alanine, glycine), might explain why some amino acids only supported slow growth of the glut-aux strain (Hubbard and Stadtman, 1967).

The possibility to modify ammonium assimilation and metabolism by metabolic engineering indicates that the underlying metabolic network is highly flexible, which is supported by highly promiscuous transaminases. However, transaminases are a limiting factor that defines the amination network, which implies the presence of different amination networks in different organisms that express different transaminases. That said, our study demonstrates how amino acid usage and even ammonium assimilation mechanisms are transferable by expressing heterologous genes. In this, we find that to allow such transferability between apparently different amination networks, the amine distribution network around the universally conserved core principle of glutamate-based amine assimilation must be highly flexible. Additionally, these engineering possibilities might have relevance for the metabolic engineering of synthetic pathways, for example, for growth-coupled selection (Orsi et al., 2021) or the production of certain amino acids or their derivatives. By engineering ammonium assimilation via non-glutamate-producing reactions, the cellular ammonium assimilation can be decoupled from glutamate biosynthesis for the production of nitrogenous compounds. Such decoupling follows the paradigm of modular design for bioengineering, which can support new endeavors for strain creation for biotechnological production processes. Here, the most obvious impact could be made on the million-ton-scale industrial amino acid production (Wendisch, 2020). Engineering new ammonium entry points as well as extending the amination network could positively affect these processes. For example, such metabolic network optimization was previously demonstrated by increased arginine production yields in Corynebacterium glutamicum upon expression of E. coli AspA and GlnA (Guo et al., 2017).

Altogether, our research addressed three distinct and complementary aspects regarding amino acid and ammonium metabolism. We provided a comprehensive overview of the options and limitations of the cellular amination network. Then, we showed its flexibility when engineering the network towards the use of new amine sources. Finally, by engineering new ammonium entry points, we increased the potential design space for engineering ammonium assimilation and dissimilation.

sequencing data has been deposited at Dryad.

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Author details

1. Helena Schulz-Mirbach

   Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany

   Contribution

   Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5376-9185

2. Alexandra Müller

   Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Tong Wu

   Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Pascal Pfister

   Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Selçuk Aslan

   Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany

   Contribution

   Supervision

   Competing interests

   No competing interests declared

6. Lennart Schada von Borzyskowski

   1. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
   2. Institute of Biology Leiden, Leiden University, Leiden, Netherlands

   Contribution

   Conceptualization, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

7. Tobias J Erb

   1. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
   2. Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

8. Arren Bar-Even

   Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany

   Contribution

   Conceptualization, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1039-4328

9. Steffen N Lindner

   1. Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany
   2. Department of Biochemistry, Charité – Universitätsmedizin Berlin, Berlin, Germany

   Contribution

   Conceptualization, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   Lindner@mpimp-golm.mpg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3226-3043

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