Biology subjects

Antoniewicz, M. R.

Publications and source records attributed to Antoniewicz, M. R..

5 recordsLinked to original sources

bioRxiv2026

The Sensitivity of 13C-Metabolic Flux Analysis to Biomass Composition Underscores the Need for Accurate Biomass Formulation in Escherichia coli

Quantifying intracellular fluxes through 13C-metabolic flux analysis (13C-MFA) provides a detailed view of cellular metabolism. While model formulation, including biomass synthesis reactions, is known to influence flux estimations, the degree to which biomass composition affects 13C-MFA outcomes in Escherichia coli remains debated. Here, we reconstructed a genome-scale 13C-MFA model of E. coli incorporating explicit nucleotide biosynthesis and a modular biomass formulation that separates protein, RNA, and other components. Using isotopic labeling experiments and systematic perturbations of biomass composition, we demonstrate that 13C-MFA can robustly differentiate subtle shifts in the protein/RNA ratio, with deviations as small as 2-3% producing statistically significant but biologically interpretable changes in model fit. Reaction-level analyses revealed consistent flux adjustments across amino acid and nucleotide biosynthesis, while pathways in one-carbon metabolism, particularly serine, glycine, and threonine interconversions, exhibited the strongest responses. Collectively, these results highlight the robustness and discriminative power of 13C-MFA and also demonstrate the importance of accurate biomass composition measurement in E. coli 13C-MFA, given the intrinsic sensitivity of the method to macromolecular stoichiometry.

systems biology↗

bioRxiv2026

Exhaustive Isotope Tracing Reveals the Butterfly Effect of Mammalian Producer Cell Line Selection for Biomanufacturing

Chinese hamster ovary (CHO) cells are the dominant platform for recombinant biotherapeutics, yet the impact of producer cell line selection on mammalian cell metabolism remains poorly understood. Here, we performed 41 independent 13C-tracer experiments using uniformly labeled glucose or individual amino acids to comprehensively map carbon utilization in the two principal CHO production platforms: methotrexate-selected CHO-K1 and glutamine synthetase-selected CHO-GS cells. Time-resolved GC-MS analysis revealed distinct metabolic phenotypes spanning central carbon metabolism, amino acid interconversion, lipid biosynthesis, and one-carbon metabolism. CHO-K1 cells exhibited extensive reductive carboxylation and pyruvate carboxylase-mediated anaplerosis, whereas CHO-GS cells redirected glutamate toward glutamine synthesis and relied on asparagine and aspartate to support TCA cycle activity. Isotopomer analysis uncovered substantial intracellular-extracellular cycling of alanine, glycine, glutamate, and serine despite contrasting uptake profiles and quantified differential amino acid contributions to fatty acids and cholesterol. Serine, glycine, and methionine labeling revealed active folate-cycle interconversion in CHO-K1 and enhanced methionine-cycle activity in CHO-GS. Aspartate is identified as a key redox exchange factor and uniquely informative tracer for pathway characterization following glutamine depletion. Together, this exhaustive isotope-tracing framework establishes how producer cell line selection rewires mammalian metabolism and provides a foundation for cell engineering, media optimization, metabolic modeling, and next-generation biomanufacturing.

cell biology↗

bioRxiv2026

Hydrogen sulfide dynamically upregulates copper uptake and localization

The reactivity of copper, an essential micronutrient that undergoes facile cycling between Cu1+ and Cu2+ redox states, is carefully controlled within the confines of protein binding sites, and by sequestration in storage vesicles, or harnessed to kill pathogens by active pumping of Cu1+ into phagosomes. We have discovered that hydrogen sulfide, a signaling metabolite generated in copious quantities at the host-microbiome interface, upregulates Cu accumulation in diffusely dispersed puncta across the cell, as visualized by X-ray fluorescence microscopy. The Cu is predominantly in the Cu2+ state with oxygen/nitrogen ligands. Cu import occurs via the non- canonical ZNT1 transporter, while export, following sulfide withdrawal, is ATP7A-dependent. Cu accumulates at the apices of colon crypts in a mouse model of elevated sulfide exposure due to SQOR deficiency in the intestinal epithelium, establishing in vivo relevance. Our study reveals that sulfide is a dynamic regulator of the Cu pool, stimulating Cu2+ influx into highly concentrated puncta.

cell biology↗

bioRxiv2025

Accelerated osteocytic citrate production in chronic kidney disease is associated with protection of the kidney

Patients with chronic kidney disease (CKD) face elevated fracture incidences, but mechanisms underlying CKD-related bone loss remain unclear. Using the adenine-induced chronic kidney injury (AdKI) murine model, we identified that AdKI induces dysregulated glucose metabolism in bones and kidneys via in vivo and ex vivo metabolic tracing. Ex vivo 13C-metabolic tracing of osteocyte-enriched femora revealed accelerated citrate production from [1,2-13C]-glucose and [U-13C]-glutamine in AdKI mice. These metabolic changes were observed together with increased circulating citrate and Slc13a5 overexpression in bones from AdKI mice. Thus, to explore the role of citrate in AdKI, we utilized mice harboring a loss of function mutation in the citrate importer SLC13A5 (Slc13a5R337*/R337*). Mutant mice displayed elevated osteocytic citrate production, and elevated circulating citrate, without significantly worsened AdKI-related bone loss. Coincident with this, Slc13a5R337*/R337* mutant mice were significantly protected from loss of kidney function with attenuated AdKI-induced nephrolithiasis. We also confirmed that Slc13a5 is highly expressed in cortical bone compared to the kidney, suggesting the effect of the mutation is mediated by SLC13A5s function outside the kidney. Altogether, this study finds that accelerated osteocytic citrate production in CKD is associated with protection of kidney function, and modulation of citrate handling may be a site for therapeutic intervention in CKD.

physiology↗

bioRxiv2020

Laboratory evolution of multiple E. coli strains reveals unifying principles of adaptation but diversity in driving genotypes

Fitness landscapes are a central concept in evolutionary biology and have been thoroughly detailed in terms of genotypes. However, our understanding of the selected metabolic and gene expression adaptations, and their dependence on genetic background, remains limited. Here, we reveal multi-scale adaptation principles in the E. coli species by taking multi-omics measurements of six different strains throughout their adaptive evolution to glucose minimal media. Statistics and matrix factorization is applied to yield four key results. First, analysis of the metabolic and physiological data shows evolutionary convergence in growth rate, glucose uptake rate, glycolytic ATP and NADH production but divergence in NADPH production strategies. Second, factorization-based analysis of the transcriptome revealed six conserved transcriptomic adaptations describing increased expression of ribosome and amino acid biosynthetic genes and decreased expression of stress response and structural genes. Third, correlation analysis identifies five tradeoffs underlying the transcriptomic profiles. Fourth, statistical tests leveraging ALE design identify four mutation-flux correlates and eight mutation-transcriptomic correlates that link mutations to systems level adaptation principles. Our total results reveal the dominant metabolic and regulatory constraints governing E. coli growth adaptation that either distinguish strains or are conserved principles.

systems biology↗