Bringing carbon to life via one-carbon metabolism.

One-carbon (C1) compounds found in greenhouse gases and industrial waste streams are underutilized carbon and energy sources. While various biological and chemical means exist for converting C1 substrates into multicarbon products, major challenges of C1 conversion lie in creating net value. Here, we review metabolic strategies to utilize carbon across oxidation states. Complications arise in biochemical C1-utilization approaches because of the need for cellular energy currency ATP. ATP supports cell maintenance and proliferation and drives thermodynamically challenging reactions by coupling them with ATP hydrolysis. Powering metabolism through substrate cofeeding and energy transduction from light and electricity improves ATP availability, relieves metabolic bottlenecks, and upcycles carbon. We present a bioenergetic, engineering, and technoeconomic outlook for bringing elements to life.

Laccase Immobilized on Arginine-Functionalized Boron Nitride Nanosheets for Enhanced Atrazine Degradation.

Enzyme-mediated systems have been widely employed for the biotransformation of environmental contaminants. However, the catalytic performance of free enzymes is restricted by the rapid loss of their catalytic activity, stability, and reusability. In this work, we developed an enzyme immobilization platform by elaborately anchoring fungal laccase onto arginine-functionalized boron nitride nanosheets (BNNS-Arg@Lac). BNNS-Arg@Lac showcased ∼75% immobilization yield and enhanced stability against fluctuating pH values and temperatures, along with remarkable reusability across six consecutive cycles, outperforming free natural laccase (nlaccase). A model pollutant, atrazine, was selected for a proof-of-concept demonstration, given the substantial environmental and public health concerns in agriculture runoff. BNNS-Arg@Lac-catalyzed atrazine degradation rate was nearly twice that of nlaccase. Moreover, BNNS-Arg@Lac consistently demonstrated superior atrazine degradation in synthetic agricultural wastewater and various mediator systems compared to nlaccase. Comprehensive product analysis unraveled distinct degradation pathways for BNNS-Arg@Lac and nlaccase. Overall, this research provides a foundation for the future development of enzyme-nanomaterial hybrids for degrading environmental chemicals and may unlock new potential for green and efficient resource recovery and waste management strategies.

Robustness of mitochondrial biogenesis and respiration explain aerobic glycolysis.

A long-standing observation is that in fast-growing cells, respiration rate declines with increasing growth rate and is compensated by an increase in fermentation, despite respiration being more efficient than fermentation. This apparent preference for fermentation even in the presence of oxygen is known as aerobic glycolysis, and occurs in bacteria, yeast, and cancer cells. Considerable work has focused on understanding the potential benefits that might justify this seemingly wasteful metabolic strategy, but its mechanistic basis remains unclear. Here we show that aerobic glycolysis results from the saturation of mitochondrial respiration and the decoupling of mitochondrial biogenesis from the production of other cellular components. Respiration rate is insensitive to acute perturbations of cellular energetic demands or nutrient supplies, and is explained simply by the amount of mitochondria per cell. Mitochondria accumulate at a nearly constant rate across different growth conditions, resulting in mitochondrial amount being largely determined by cell division time. In contrast, glucose uptake rate is not saturated, and is accurately predicted by the abundances and affinities of glucose transporters. Combining these models of glucose uptake and respiration provides a quantitative, mechanistic explanation for aerobic glycolysis. The robustness of specific respiration rate and mitochondrial biogenesis, paired with the flexibility of other bioenergetic and biosynthetic fluxes, may play a broad role in shaping eukaryotic cell metabolism.

Integrated Proteomics and Metabolomics Reveal Altered Metabolic Regulation of Xanthobacter autotrophicus under Electrochemical Water-Splitting Conditions.

Biological-inorganic hybrid systems are a growing class of technologies that combine microorganisms with materials for a variety of purposes, including chemical synthesis, environmental remediation, and energy generation. These systems typically consider microorganisms as simple catalysts for the reaction of interest; however, other metabolic activity is likely to have a large influence on the system performance. The investigation of biological responses to the hybrid environment is thus critical to the future development and optimization. The present study investigates this phenomenon in a recently reported hybrid system that uses electrochemical water splitting to provide reducing equivalents to the nitrogen-fixing bacteria Xanthobacter autotrophicus for efficient reduction of N2 to biomass that may be used as fertilizer. Using integrated proteomic and metabolomic methods, we find a pattern of differentiated metabolic regulation under electrochemical water-splitting (hybrid) conditions with an increase in carbon fixation products glycerate-3-phosphate and acetyl-CoA that suggests a high energy availability. We further report an increased expression of proteins of interest, namely, those responsible for nitrogen fixation and assimilation, which indicate increased rates of nitrogen fixation and support previous observations of faster biomass accumulation in the hybrid system compared to typical planktonic growth conditions. This work complicates the inert catalyst view of biological-inorganic hybrids while demonstrating the power of multiomics analysis as a tool for deeper understanding of those systems.

Transcriptomic characteristics according to tumor size and SUVmax in papillary thyroid cancer patients.

The SUVmax is a measure of FDG uptake and is related with tumor aggressiveness in thyroid cancer, however, its association with molecular pathways is unclear. Here, we investigated the relationship between SUVmax and gene expression profiles in 80 papillary thyroid cancer (PTC) patients. We conducted an analysis of DEGs and enriched pathways in relation to SUVmax and tumor size. SUVmax showed a positive correlation with tumor size and correlated with glucose metabolic process. The genes that indicate thyroid differentiation, such as SLC5A5 and TPO, were negatively correlated with SUVmax. Unsupervised analysis revealed that SUVmax positively correlated with DNA replication(r = 0.29, p = 0.009), pyrimidine metabolism(r = 0.50, p < 0.0001) and purine metabolism (r = 0.42, p = 0.0001). Based on subgroups analysis, we identified that PSG5, TFF3, SOX2, SL5A5, SLC5A7, HOXD10, FER1L6, and IFNA1 genes were found to be significantly associated with tumor aggressiveness. Both high SUVmax PTMC and macro-PTC are enriched in pathways of DNA replication and cell cycle, however, gene sets for purine metabolic pathways are enriched only in high SUVmax macro-PTC but not in high SUVmax PTMC. Our findings demonstrate the molecular characteristics of high SUVmax tumor and metabolism involved in tumor growth in differentiated thyroid cancer.

Neutrophil diversity is associated with T-cell immunity and clinical relevance in patients with thyroid cancer.

Neutrophil heterogeneity is involved in autoimmune diseases, sepsis, and several cancers. However, the link between neutrophil heterogeneity and T-cell immunity in thyroid cancer is incompletely understood. We investigated the circulating neutrophil heterogeneity in 3 undifferentiated thyroid cancer (UTC), 14 differentiated thyroid cancer (DTC) (4 Stage IV, 10 Stage I-II), and healthy controls (n = 10) by transcriptomic data and cytometry. Participants with UTC had a significantly higher proportion of immature high-density neutrophils (HDN) and lower proportion of mature HDN in peripheral blood compared to DTC. The proportion of circulating PD-L1+ immature neutrophils were significantly increased in advanced cancer patients. Unsupervised analysis of transcriptomics data from circulating HDN revealed downregulation of innate immune response and T-cell receptor signaling pathway in cancer patients. Moreover, UTC patients revealed the upregulation of glycolytic process and glutamate receptor signaling pathway. Comparative analysis across tumor types and stages revealed the downregulation of various T-cell-related pathways, such as T-cell receptor signaling pathway and T-cell proliferation in advanced cancer patients. Moreover, the proportions of CD8+ and CD4+ T effector memory CD45RA+ (TEMRA) cells from peripheral blood were significantly decreased in UTC patients compared to DTC patients. Finally, we demonstrated that proportions of tumor-infiltrated neutrophils were increased and related with poor prognosis in advanced thyroid cancer using data from our RNA-seq and TCGA (The Cancer Genome Atlas) data. In conclusion, observed prevalence of circulating immature high-density neutrophils and their immunosuppressive features in undifferentiated thyroid cancers underscore the importance of understanding neutrophil dynamics in the context of tumor progression in thyroid cancer.

Extracellular domains of CARs reprogramme T cell metabolism without antigen stimulation.

Metabolism is an indispensable part of T cell proliferation, activation and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are composed of extracellular domains-often single-chain variable fragments (scFvs)-that determine ligand specificity and intracellular domains that trigger signalling following antigen binding. Here, we show that CARs differing only in the scFv variously reprogramme T cell metabolism. Even without exposure to antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observed basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harbouring a rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14G2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer cells and CAR-T cells are identified as features of efficacious CAR-T cell therapy.

Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses.

The role of the serine/glycine metabolic pathway (SGP) has recently been demonstrated in tumors; however, the pathological relevance of the SGP in thyroid cancer remains unexplored. Here, we perform metabolomic profiling of 17 tumor-normal pairs; bulk transcriptomics of 263 normal thyroid, 348 papillary, and 21 undifferentiated thyroid cancer samples; and single-cell transcriptomes from 15 cases, showing the impact of mitochondrial one-carbon metabolism in thyroid tumors. High expression of serine hydroxymethyltransferase-2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is associated with low thyroid differentiation scores and poor clinical features. A subpopulation of tumor cells with high mitochondrial one-carbon pathway activity is observed in the single-cell dataset. SHMT2 inhibition significantly compromises mitochondrial respiration and decreases cell proliferation and tumor size in vitro and in vivo. Collectively, our results highlight the importance of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer and suggest that SHMT2 is a potent therapeutic target.

A parallel glycolysis provides a selective advantage through rapid growth acceleration.

Glycolysis is a universal metabolic process that breaks down glucose to produce adenosine triphosphate (ATP) and biomass precursors. The Entner-Doudoroff (ED) pathway is a glycolytic pathway that parallels textbook glycolysis but yields half as much ATP. Accordingly, in organisms that possess both glycolytic pathways (for example, Escherichia coli), its raison d'être remains a mystery. In this study, we found that the ED pathway provides a selective advantage during growth acceleration. Upon carbon and nitrogen upshifts, E. coli accelerates growth faster with than without the ED pathway. Concurrent isotope tracing reveals that the ED pathway flux increases faster than that of textbook glycolysis. We attribute the fast response time of the ED pathway to its strong thermodynamic driving force and streamlining of glucose import. Intermittent nutrient supply manifests the evolutionary advantage of the parallel glycolysis; thus, the dynamic nature of an ostensibly redundant pathway's role in promoting rapid adaptation constitutes a metabolic design principle.

Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy.

Mounting evidence suggests metabolism instructs stem cell fate decisions. However, how fetal metabolism changes during development and how altered maternal metabolism shapes fetal metabolism remain unexplored. We present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and liquid chromatography-mass spectrometry (LC-MS), we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Our analysis revealed metabolic features specific to a hyperglycemic environment and signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from hyperglycemic dams. Tracing 13C-glucose revealed disparate fetal nutrient sourcing depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated in late-stage fetal tissues. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations due to maternal hyperglycemia.

Determination of Metabolic Fluxes by Deep Learning of Isotope Labeling Patterns.

Fluxomics offers a direct readout of metabolic state but relies on indirect measurement. Stable isotope tracers imprint flux-dependent isotope labeling patterns on metabolites we measure; however, the relationship between labeling patterns and fluxes remains elusive. Here we innovate a two-stage machine learning framework termed ML-Flux that streamlines metabolic flux quantitation from isotope tracing. We train machine learning models by simulating atom transitions across five universal metabolic models starting from 26 13C-glucose, 2H-glucose, and 13C-glutamine tracers within feasible flux space. ML-Flux employs deep-learning-based imputation to take variable measurements of labeling patterns as input and successive neural networks to convert the ensuing comprehensive labeling information into metabolic fluxes. Using ML-Flux with multi-isotope tracing, we obtain fluxes through central carbon metabolism that are comparable to those from a least-squares method but orders-of-magnitude faster. ML-Flux is deployed as a webtool to expand the accessibility of metabolic flux quantitation and afford actionable information on metabolism.

Toward Systems-Level Metabolic Analysis in Endocrine Disorders and Cancer.

Metabolism is a dynamic network of biochemical reactions that support systemic homeostasis amidst changing nutritional, environmental, and physical activity factors. The circulatory system facilitates metabolite exchange among organs, while the endocrine system finely tunes metabolism through hormone release. Endocrine disorders like obesity, diabetes, and Cushing's syndrome disrupt this balance, contributing to systemic inflammation and global health burdens. They accompany metabolic changes on multiple levels from molecular interactions to individual organs to the whole body. Understanding how metabolic fluxes relate to endocrine disorders illuminates the underlying dysregulation. Cancer is increasingly considered a systemic disorder because it not only affects cells in localized tumors but also the whole body, especially in metastasis. In tumorigenesis, cancer-specific mutations and nutrient availability in the tumor microenvironment reprogram cellular metabolism to meet increased energy and biosynthesis needs. Cancer cachexia results in metabolic changes to other organs like muscle, adipose tissue, and liver. This review explores the interplay between the endocrine system and systems-level metabolism in health and disease. We highlight metabolic fluxes in conditions like obesity, diabetes, Cushing's syndrome, and cancers. Recent advances in metabolomics, fluxomics, and systems biology promise new insights into dynamic metabolism, offering potential biomarkers, therapeutic targets, and personalized medicine.

Unexpected metabolic rewiring of CO2 fixation in H2-mediated materials-biology hybrids.

A hybrid approach combining water-splitting electrochemistry and H2-oxidizing, CO2-fixing microorganisms offers a viable solution for producing value-added chemicals from sunlight, water, and air. The classic wisdom without thorough examination to date assumes that the electrochemistry in such a H2-mediated process is innocent of altering microbial behavior. Here, we report unexpected metabolic rewiring induced by water-splitting electrochemistry in H2-oxidizing acetogenic bacterium Sporomusa ovata that challenges such a classic view. We found that the planktonic S. ovata is more efficient in utilizing reducing equivalent for ATP generation in the materials-biology hybrids than cells grown with H2 supply, supported by our metabolomic and proteomic studies. The efficiency of utilizing reducing equivalents and fixing CO2 into acetate has increased from less than 80% of chemoautotrophy to more than 95% under electroautotrophic conditions. These observations unravel previously underappreciated materials' impact on microbial metabolism in seemingly simply H2-mediated charge transfer between biotic and abiotic components. Such a deeper understanding of the materials-biology interface will foster advanced design of hybrid systems for sustainable chemical transformation.

Sustainable metabolic engineering requires a perfect trifecta.

The versatility of cellular metabolism in converting various substrates to products inspires sustainable alternatives to conventional chemical processes. Metabolism can be engineered to maximize the yield, rate, and titer of product generation. However, the numerous combinations of substrate, product, and organism make metabolic engineering projects difficult to navigate. A perfect trifecta of substrate, product, and organism is prerequisite for an environmentally and economically sustainable metabolic engineering endeavor. As a step toward this endeavor, we propose a reverse engineering strategy that starts with product selection, followed by substrate and organism pairing. While a large bioproduct space has been explored, the top-ten compounds have been synthesized mainly using glucose and model organisms. Unconventional feedstocks (e.g. hemicellulosic sugars and CO2) and non-model organisms are increasingly gaining traction for advanced bioproduct synthesis due to their specialized metabolic modes. Judicious selection of the substrate-organism-product combination will illuminate the untapped territory of sustainable metabolic engineering.

M2 isoform of pyruvate kinase rewires glucose metabolism during radiation therapy to promote an antioxidant response and glioblastoma radioresistance.

BACKGROUND: Resistance to existing therapies is a significant challenge in improving outcomes for glioblastoma (GBM) patients. Metabolic plasticity has emerged as an important contributor to therapy resistance, including radiation therapy (RT). Here, we investigated how GBM cells reprogram their glucose metabolism in response to RT to promote radiation resistance. METHODS: Effects of radiation on glucose metabolism of human GBM specimens were examined in vitro and in vivo with the use of metabolic and enzymatic assays, targeted metabolomics, and FDG-PET. Radiosensitization potential of interfering with M2 isoform of pyruvate kinase (PKM2) activity was tested via gliomasphere formation assays and in vivo human GBM models. RESULTS: Here, we show that RT induces increased glucose utilization by GBM cells, and this is accompanied with translocation of GLUT3 transporters to the cell membrane. Irradiated GBM cells route glucose carbons through the pentose phosphate pathway (PPP) to harness the antioxidant power of the PPP and support survival after radiation. This response is regulated in part by the PKM2. Activators of PKM2 can antagonize the radiation-induced rewiring of glucose metabolism and radiosensitize GBM cells in vitro and in vivo. CONCLUSIONS: These findings open the possibility that interventions designed to target cancer-specific regulators of metabolic plasticity, such as PKM2, rather than specific metabolic pathways, have the potential to improve the radiotherapeutic outcomes in GBM patients.

Extracellular Domains of CAR Reprogram T-Cell Metabolism Without Antigen Stimulation.

Metabolism is an indispensable part of T-cell proliferation, activation, and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are comprised of extracellular domains that determine cancer specificity, often using single-chain variable fragments (scFvs), and intracellular domains that trigger signaling upon antigen binding. Here we show that CARs differing only in the scFv reprogram T-cell metabolism differently. Even in the absence of antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observe basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harboring rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14g2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Since CAR-dependent metabolic reprogramming alters cellular energetics, nutrient utilization, and proliferation, metabolic profiling should be an integral part of CAR-T cell development.

Integrative metabolic flux analysis reveals an indispensable dimension of phenotypes.

Complete understanding of a biological system requires quantitation of metabolic fluxes that reflect its dynamic state. Various analytical chemistry tools, enzyme-based probes, and microscopy enable flux measurement. However, any method alone falls short of comprehensive flux quantitation. Here we show that integrating these techniques results in a systems-level quantitative map of absolute metabolic fluxes that constitute an indispensable dimension of characterizing phenotypes. Stable isotopes, mass spectrometry, and NMR spectroscopy reveal relative pathway fluxes. Biochemical probes reveal the physical rate of environmental changes. FRET-based and SRS-based microscopy reveal targeted metabolite and chemical bond formation. These techniques are complementary and can be computationally integrated to reveal actionable information on metabolism. Integrative metabolic flux analysis using various quantitative techniques advances biotechnology and medicine.

Maximizing light-driven CO2 and N2 fixation efficiency in quantum dot-bacteria hybrids.

Integrating light-harvesting materials with microbial biochemistry is a viable approach to produce chemicals with high efficiency from the air, water, and sunlight. Yet it remains unclear whether all absorbed photons in the materials can be transferred through the material-biology interface for solar-to-chemical production and whether the presence of materials beneficially affect the microbial metabolism. Here we report a microbe-semiconductor hybrid by interfacing CO2/N2-fixing bacterium Xanthobacter autotrophicus with CdTe quantum dots for light-driven CO2 and N2 fixation with internal quantum efficiencies of 47.2 ± 7.3% and 7.1 ± 1.1%, respectively, reaching the biochemical limits of 46.1% and 6.9% imposed by the stoichiometry in biochemical pathways. Photophysical studies suggest fast charge-transfer kinetics at the microbe-semiconductor interfaces, while proteomics and metabolomics indicate a material-induced regulation of microbial metabolism favoring higher quantum efficiencies compared to the biological counterparts alone.

Light-Independent Biological Conversion of CO2.

Sevcan Ersan is a postdoctoral researcher at UCLA. Previously, she conducted postdoctoral research at the University of Hohenheim in Germany. She received her PhD in biotechnology from Yeditepe University, Turkey, and her bachelor's and master's degrees in food engineering from Istanbul Technical University, Turkey. She is experienced in waste utilization, bioprocessing technologies, and biological activities associated with phytochemicals. Her current research focuses on natural product chemistry and sustainable biotechnology. Junyoung Park is an assistant professor of Chemical and Biomolecular Engineering and co-director of the Metabolomics Center at UCLA. His research group focuses on systems-level analysis of metabolic networks to elucidate regulatory mechanisms and engineer metabolism. He aims to apply this knowledge to solving energy and environmental problems and curing human diseases such as cancer and diabetes. Before moving to Los Angeles, he conducted postdoctoral research at MIT. He received his bachelor's degrees in mathematics and bioengineering from UC San Diego and a master's and PhD in chemical engineering from Princeton University.