Glycine Pathway Research Articles

Long-Term Autotrophic Growth and Solar-to-Chemical Conversion in Shewanella Oneidensis MR-1 through Light-Driven Electron Transfer.

Members of the genus Shewanella are known for their versatile electron accepting routes, which allow them to couple decomposition of organic matter to reduction of various terminal electron acceptors for heterotrophic growth in diverse environments. Here, we report autotrophic growth of Shewanella oneidensis MR-1 with photoelectrons provided by illuminated biogenic CdS nanoparticles. This hybrid system enables photosynthetic oscillatory acetate production from CO2 for over five months, far exceeding other inorganic-biological hybrid system that can only sustain for hours or days. Biochemical, electrochemical and transcriptomic analyses reveal that the efficient electron uptake of S. oneidensis MR-1 from illuminated CdS nanoparticles supplies sufficient energy to stimulate the previously overlooked reductive glycine pathway for CO2 fixation. The continuous solar-to-chemical conversion is achieved by photon induced electric recycling in sulfur species. Overall, our findings demonstrate that this mineral-assisted photosynthesis, as a widely existing and unique model of light energy conversion, could support the sustained photoautotrophic growth of non-photosynthetic microorganisms in nutrient-lean environments and mediate the reversal of coupled carbon and sulfur cycling, consequently resulting in previously unknown environmental effects. In addition, the hybrid system provides a sustainable and flexible platform to develop a variety of solar products for carbon neutrality.

Long‐Term Autotrophic Growth and Solar‐to‐Chemical Conversion in Shewanella Oneidensis MR‐1 through Light‐Driven Electron Transfer

AbstractMembers of the genus Shewanella are known for their versatile electron accepting routes, which allow them to couple decomposition of organic matter to reduction of various terminal electron acceptors for heterotrophic growth in diverse environments. Here, we report autotrophic growth of Shewanella oneidensis MR‐1 with photoelectrons provided by illuminated biogenic CdS nanoparticles. This hybrid system enables photosynthetic oscillatory acetate production from CO2 for over five months, far exceeding other inorganic‐biological hybrid system that can only sustain for hours or days. Biochemical, electrochemical and transcriptomic analyses reveal that the efficient electron uptake of S. oneidensis MR‐1 from illuminated CdS nanoparticles supplies sufficient energy to stimulate the previously overlooked reductive glycine pathway for CO2 fixation. The continuous solar‐to‐chemical conversion is achieved by photon induced electric recycling in sulfur species. Overall, our findings demonstrate that this mineral‐assisted photosynthesis, as a widely existing and unique model of light energy conversion, could support the sustained photoautotrophic growth of non‐photosynthetic microorganisms in nutrient‐lean environments and mediate the reversal of coupled carbon and sulfur cycling, consequently resulting in previously unknown environmental effects. In addition, the hybrid system provides a sustainable and flexible platform to develop a variety of solar products for carbon neutrality.

Multi-scale reactor designs extend the physical limits of fixation.

valorization is a promising strategy for climate adaptation and transitioning towards a circular carbon economy. Here, we present a multi-scale, integrated systems approach for designing biomanufacturing systems that can utilize as a feedstock, focusing on the Wood-Ljungdahl and reductive glycine pathways. This approach relies on first principles, coupling the optimization of pathway and process variables. We examine the -fixation capacity of both pathways in single- and multi-compartment reactor systems, demonstrating that the reductive glycine pathway has the potential to fix at significantly higher rates than photosynthetic organisms. We show that small differences in the energy-dissipative and stoichiometric structures of carbon-fixation pathways could significantly impact optimal designs and feasible design spaces. Our first-principle, systems-level approach quantifies these differences and uncovers strategies to expand the design space and extend the physical limits of carbon fixation, offering insights into pathway selection and process configurations for efficient biomanufacturing.

Cultivation of novel Atribacterota from oil well provides new insight into their diversity, ecology, and evolution in anoxic, carbon-rich environments

BackgroundThe Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood.ResultsWe report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments.ConclusionOur findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory.5rnPv2dUCRDw24_qsw9GBLVideo

Engineering yeasts to Co-utilize methanol or formate coupled with CO2 fixation

The development of synthetic microorganisms that could use one-carbon compounds, such as carbon dioxide, methanol, or formate, has received considerable interest. In this study, we engineered Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotrophy and formatotrophy, enabling them to co-utilize methanol or formate with CO2 fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consisted of a methanol-formate oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using endogenous enzymes, followed by blocking the native methanol assimilation pathway, modularly engineering genes of MFORG pathway, and compartmentalizing the methanol oxidation module. These modifications successfully enabled the methylotrophic yeast P. pastoris to utilize both methanol and formate. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing the synthetic methylotrophy and formatotrophy in this organism. The resulting strain could also successfully utilize both methanol and formate with consumption rates of 20 mg/L/h and 36.5 mg/L/h, respectively. The ability of the engineered P. pastoris and S. cerevisiae to co-assimilate CO2 with methanol or formate through the MFORG pathway was also confirmed by 13C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid by co-assimilating methanol and CO2 was demonstrated in the engineered P. pastoris and S. cerevisiae. This work indicates the potential of the MFORG pathway in developing different hosts to use various one-carbon compounds for chemical production.

Transcriptomic and metabolomic insights into the antimony stress response of tall fescue (Festuca arundinacea)

Antimony (Sb) is a toxic heavy metal that severely inhibits plant growth and development and threatens human health. Tall fescue, one of the most widely used grasses, has been reported to tolerate heavy metal stress. However, the adaptive mechanisms of Sb stress in tall fescue remain largely unknown. In this study, transcriptomic and metabolomic techniques were applied to elucidate the molecular mechanism of the Sb stress response in tall fescue. These results showed that the defense process in tall fescue was rapidly triggered during the early stages of Sb stress. Sb stress had toxic effects on tall fescue, and the cell wall and voltage-gated channels are crucial for regulating Sb permeation into the cells. In addition, the pathway of glycine, serine and threonine metabolism may play key roles in the Sb stress response of tall fescue. Genes such as ALDH7A1 and AGXT2 and metabolites such as aspartic acid, pyruvic acid, and biuret, which are related to biological processes and pathways, were key genes and compounds in the Sb stress response of tall fescue. Therefore, the regulatory mechanisms of specific genes and pathways should be investigated further to improve Sb stress tolerance.

Interspecies cooperation-driven photogenerated electron transfer processes and efficient multi-pathway nitrogen removal in the g-C3N4-anammox consortia biohybrid system

Photocatalytic materials-microbial biohybrid systems pave the way for solar-driven wastewater nitrogen removal. In this study, interspecies cooperation in photogenerated electron transfer and efficient nitrogen removal mechanism in the g-C3N4-anammox consortia biohybrid system were first deciphered. The results indicated that the essential extracellular electron carriers (cytochrome c and flavin) for anammox genomes were provided by associated bacteria (BACT3 and CHLO2). This cooperation, regulated by the ArcAB system and electron transfer flavoprotein, made anammox bacteria the primary photogenerated electron sink. Furthermore, an efficient photogenerated electron harness was used to construct a reductive glycine pathway (rGlyP) in anammox bacteria inventively, which coexisted with the Wood–Ljungdahl pathway (WLP), constituting a dual-pathway carbon fixation model, rGlyP-WLP. Carbon fixation products efficiently contributed to the tricarboxylic acid cycle, while inhibiting electron diversion in anabolism. Photogenerated electrons were targeted channeled into nitrogen metabolism-available electron carriers, enhancing anammox and dissimilatory nitrate reduction to ammonium (DNRA) processes. Moreover, ammonia assimilation by the glycine cleavage system in rGlyP established an alternative ammonia removal route. Ultimately, multi-pathway nitrogen removal involving anammox, DNRA, and rGlyP achieved 100 % ammonia removal and 94.25 % total nitrogen removal efficiency. This study has expanded understanding of anammox metabolic diversity, enhancing its potential application in carbon-neutral wastewater treatment.

Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO2 assimilation

Using captured CO2 and C1-feedstocks like formate and methanol derived from electrochemical activation of CO2 are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO2-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO2 assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO2. ALE improved the RGP-dependent growth by 8-fold. 13C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we re-established formate and CO2-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more 13C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO2 assimilation.

The oxygen-tolerant reductive glycine pathway assimilates methanol, formate and CO2 in the yeast Komagataella phaffii

The current climatic change is predominantly driven by excessive anthropogenic CO2 emissions. As industrial bioprocesses primarily depend on food-competing organic feedstocks or fossil raw materials, CO2 co-assimilation or the use of CO2-derived methanol or formate as carbon sources are considered pathbreaking contributions to solving this global problem. The number of industrially-relevant microorganisms that can use these two carbon sources is limited, and even fewer can concurrently co-assimilate CO2. Here, we search for alternative native methanol and formate assimilation pathways that co-assimilate CO2 in the industrially-relevant methylotrophic yeast Komagataella phaffii (Pichia pastoris). Using 13C-tracer-based metabolomic techniques and metabolic engineering approaches, we discover and confirm a growth supporting pathway based on native enzymes that can perform all three assimilations: namely, the oxygen-tolerant reductive glycine pathway. This finding paves the way towards metabolic engineering of formate and CO2 utilisation to produce proteins, biomass, or chemicals in yeast.

Electrical-biological hybrid system for carbon efficient isobutanol production

We have developed an electrical-biological hybrid system wherein an engineered microorganism consumes electrocatalytically produced formate from CO2 to supplement the bioproduction of isobutanol, a valuable fuel chemical. Biological CO2 sequestration is notoriously slow compared to electrochemical CO2 reduction, while electrochemical methods struggle to generate carbon-carbon bonds which readily form in biological systems. A hybrid system provides a promising method for combining the benefits of both biology and electrochemistry. Previously, Escherichia coli was engineered to assimilate formate and CO2 in central metabolism using the reductive glycine pathway. In this work, we have shown that chemical production in E. coli can benefit from single carbon substrates when equipped with the RGP. By installing the RGP and the isobutanol biosynthetic pathway into E. coli and by further genetic modifications, we have generated a strain of E. coli that can consume formate and produce isobutanol at a yield of >100% of theoretical maximum from glucose. Our results demonstrate that carbon produced from electrocatalytically reduced CO2 can bolster chemical production in E. coli. This study shows that E. coli can be engineered towards carbon efficient methods of chemical production.

Effect of temperature on the degradation of glyphosate by Mn-oxide: Products and pathways of degradation

Glyphosate is the most commonly used herbicide in the United States. In the environment, glyphosate residues can either degrade into more toxic and persistent byproducts such as aminomethylphosphonic acid (AMPA) or environmentally benign species such as sarcosine or glycine. In this research, the birnessite-catalyzed degradation of glyphosate was studied under environmentally relevant temperatures (10–40 °C) using high-performance liquid chromatography, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and theoretical calculations. Our results show a temperature-dependent degradation pathway preference for AMPA and glycine production. The AMPA and glycine pathways are competitive at short reaction times, but the glycine pathway became increasingly preferred as reaction time and temperature increased. The measured free energy barriers are comparable for both the glycine and AMPA pathways (93.5 kJ mol−1 for glycine and 97.1 kJ mol−1 for AMPA); however, the entropic energy penalty for the AMPA pathway is significantly greater than the glycine pathway (-TΔS‡ = 26.2 and 42.8 kJ mol−1 for glycine and AMPA, respectively). These findings provide possible routes for biasing glyphosate degradation towards safer products, thus to decrease the overall environmental toxicity.

Microbial Conversion and Utilization of CO2

Rising greenhouse gas emissions have contributed to unprecedented levels of climate change, while microbial conversion and utilization of CO2 is a practical way to reduce emissions and promote green manufacturing. This article mainly summarizes several natural CO2 pathways that have been discovered, including the Calvin cycle, the reduced tricarboxylic acid (rTCA) cycle, the Wood–Ljungdahl (WL) pathway, the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle, the 3-hydroxypropionate (3HP) cycle, the reductive glycine (rGly) pathway, and artificially designed carbon fixation pathways includes the CETCH cycle, the MOG pathway, the acetyl-CoA bicycle, and the POAP cycle. We also discussed applications of different carbon fixation enzymes, notably ribulose-1, 5-diphosphate carboxylase/oxygenase, pyruvate carboxylase, carbonic anhydrase, as well as formate dehydrogenase. This paper further addressed the development of photosynthetic autotrophs, chemergic autotrophs and model bacteria Escherichia coli or yeast produced main products for CO2 fixation through metabolic engineering, such as alcohols, organic acids, fatty acids and lipids, bioplastics, terpenoids, hydrocarbons, and biomass. Future studies on CO2 microbial conversion should focus on improving the efficiency of carbon fixation enzymes, metabolic modules of the carbon sequestration pathway, and intracellular energy utilization. Coupled microbial and electrochemical methods for CO2 fixation, in addition to biological fixation, show considerable promise.

Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

Metabolic Versatility of the Family Halieaceae Revealed by the Genomics of Novel Cultured Isolates.

The family Halieaceae (OM60/NOR5 clade) is a gammaproteobacterial group abundant and cosmopolitan in coastal seawaters and plays an important role in response to phytoplankton blooms. However, the ecophysiology of this family remains understudied because of the vast gap between phylogenetic diversity and cultured representatives. Here, using six pure cultured strains isolated from coastal seawaters, we performed in-depth genomic analyses to provide an overview of the phylogeny and metabolic capabilities of this family. The combined analyses of 16S rRNA genes, genome sequences, and functional genes relevant to taxonomy demonstrated that each strain represents a novel species. Notably, two strains belonged to the hitherto-uncultured NOR5-4 and NOR5-12 subclades. Metabolic reconstructions revealed that the six strains likely have aerobic chemo- or photoheterotrophic lifestyles; five of them possess genes for proteorhodopsin or aerobic anoxygenic phototrophy. The presence of blue- or green-tuned proteorhodopsin in Halieaceae suggested their ability to adapt to light conditions varying with depth or coastal-to-open ocean transition. In addition to the genes of anaplerotic CO2 fixation, genes encoding a complete reductive glycine pathway for CO2 fixation were found in three strains. Putative polysaccharide utilization loci were detected in three strains, suggesting the association with phytoplankton blooms. Read mapping of various metagenomes and metatranscriptomes showed that the six strains are widely distributed and transcriptionally active in marine environments. Overall, the six strains genomically characterized in this study expand the phylogenetic and metabolic diversity of Halieaceae and likely serve as a culture resource for investigating the ecophysiological features of this environmentally relevant bacterial group. IMPORTANCE Although the family Halieaceae (OM60/NOR5 clade) is an abundant and cosmopolitan clade widely found in coastal seas and involved in interactions with phytoplankton, a limited number of cultured isolates are available. In this study, we isolated six pure cultured Halieaceae strains from coastal seawaters and performed a comparative physiological and genomic analysis to give insights into the phylogeny and metabolic potential of this family. The cultured strains exhibited diverse metabolic potential by harboring genes for anaplerotic CO2 fixation, proteorhodopsin, and aerobic anoxygenic phototrophy. Polysaccharide utilization loci detected in some of these strains also indicated an association with phytoplankton blooms. The cultivation of novel strains of Halieaceae and their genomic characteristics largely expanded the phylogenetic and metabolic diversity, which is important for future ecophysiological studies.

Rücktitelbild: Enzymatic Electrosynthesis of Glycine from CO2 and NH3 (Angew. Chem. 14/2023)

An enzymatic electrocatalytic system was developed to allow the simultaneous conversion of CO2 and NH3 for glycine synthesis, as reported by Zhiguang Zhu et al. in their Research Article (DOI: 10.1002/ange.202218387). This system was inspired by the reductive glycine pathway in vivo and based on the in vitro multienzymatic cascade reaction, representing a novel alternative of the enzymatic CO2 electroreduction for synthesizing nitrogenous value-added chemicals.

Back Cover: Enzymatic Electrosynthesis of Glycine from CO2 and NH3 (Angew. Chem. Int. Ed. 14/2023)

An enzymatic electrocatalytic system was developed that allows the simultaneous conversion of CO2 and NH3 for glycine synthesis, as reported by Zhiguang Zhu et al. in their Research Article (e202218387). This system was inspired by the reductive glycine pathway in vivo and is based on an in vitro multienzymatic cascade reaction, representing a novel alternative for enzymatic CO2 electroreduction for synthesizing nitrogenous value-added chemicals.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3

AbstractEnzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2. However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value‐added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L−1 h−1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3.

Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Paving the way for synthetic C1 - Metabolism in Pseudomonas putida through the reductive glycine pathway

One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to modularly assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module in the auxotrophic strains from formate to methanol-dependent growth using both NAD and PQQ-dependent methanol dehydrogenases. Finally, we demonstrated, for the first time, engineered CO2-dependent formation of part of the biomass through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD + ratio. This research paves the way to further engineer P. putida towards full growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells.

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo.