ABSTRACT Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13 C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L -1 muconate at 1.1 g L -1 h -1 . Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

Bacteria regulate homeostatic growth by adjusting proteome composition. In Escherichia coli, this coordination is mediated by guanosine tetraphosphate and pentaphosphate, collectively termed (p)ppGpp, which couple amino acid supply with ribsosome production. We identified a distinct architecture in Bacillus subtilis, in which guanosine triphosphate (GTP), not (p)ppGpp, controls proteome allocation. Translational inhibition resulted in GTP depletion and suppressed amino acid biosynthesis through feedback inhibition without altering ribosome abundance, establishing a regulated decoupling between total amino acid flux and proteome composition, with flux deviating from proteome-based predictions. By artificially adjusting GTP concentrations, we recoupled flux and proteome, restoring growth to maximal amounts. The regulated suboptimality enables a trade-off to balance growth and stress resilience. Similar GTP-based strategies were present in other Firmicute species, indicating possible evolutionary conservation. Proteome composition and metabolic flux have distinct regulatory layers in some bacteria.

The transition from unicellular to multicellular growth requires diversification of cellular functions within genetically identical populations. In Bacillus subtilis, biofilm formation is historically viewed as a developmental precursor to sporulation along a linear pathway. Here, we show that biofilm formation and sporulation instead diverge along a branched pathway. A subpopulation that first initiates sporulation catabolizes lipoteichoic acid through the sequential action of the enzymes ShfP (Sporulation heterogeneity factor Poison) and PhoA (alkaline phosphatase A), leading to the release of millimolar concentrations of glycerol. This glycerol impedes sporulation by disrupting cell wall synthesis and cytoplasmic pH, necessitating counteraction by another protein, ShfA (Sporulation heterogeneity factor Antidote). The extracellular glycerol, however, acts as a morphogen that directs neighboring cells to initiate biofilm formation, which we directly visualize in developing populations of cells. Thus, B. subtilis multicellularity emerges through a branched developmental program in which sporulating cells generate the cue that creates the biofilm-producing lineage via cell-cell communication through repurposing of a canonical intracellular metabolite.

Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n-butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n-butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n-butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum: a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols. IMPORTANCEThis study identifies key mechanisms of Clostridium thermocellum membrane remodeling under alcohol stress, showing that AdhE mediates incorporation of exogenous alcohols into fatty acids, while Pfor4 drives branched-chain fatty acid synthesis in the absence of the canonical Bkd complex. These findings highlight actionable targets for metabolic engineering to enhance solvent tolerance and improve the robustness and productivity of C. thermocellum as a biofuel-producing platform.

Microbial strains engineered for high-titer ethanol production often stop fermenting while substantial substrate remains, limiting industrial performance. We investigated this limitation in engineered strains of Escherichia coli and Thermoanaerobacterium saccharolyticum and the native ethanologen Zymomonas mobilis. By combining high-titer fermentations with intracellular metabolomics, we are able to see how intracellular metabolite concentrations change as product formation stops. We then used max-min driving force (MDF) thermodynamic analysis to understand how these changes in intracellular metabolite levels can limit flux and to identify key enzymes that might be responsible for these limitations. In engineered strains, cessation of ethanol production coincided with strong pyruvate accumulation and MDF values near or below zero at the pyruvate kinase step, implying that the pyruvate consuming enzyme(s) (pyruvate decarboxylase for E. coli and pyruvate ferredoxin oxidoreductase, or associated electron transfer enzymes for T. saccharolyticum) might limit flux. By contrast, Z. mobilis maintained positive driving forces without pyruvate buildup, suggesting that its titer is limited by processes outside central carbon metabolism, such as substrate uptake. These results establish a generalizable framework linking metabolite concentrations to pathway thermodynamics and demonstrate how thermodynamic analysis can diagnose where metabolic constraints emerge during high-titer fermentation.IMPORTANCEHigh-titer fermentation is essential for economically viable biofuel production, yet even extensively-engineered microbes frequently stop producing ethanol before the substrate is exhausted. Furthermore, the causes of titer limitations are often poorly understood. A particular challenge is identifying the location of titer limitations in multi-enzyme pathways. Here, we show that MDF analysis can assist in the interpretation of metabolomic data. These findings provide a systems-level explanation for "stuck" fermentations in bacteria and identify thermodynamic driving force as a quantitative diagnostic metric that reveals where biological design targets emerge for metabolic engineering of ethanol and other bioproducts.

Through biochemical transformation of host-derived bile acids, gut bacteria mediate host-microbe crosstalk and function at the interface of nutrition and host metabolic regulation. Bile acids play a crucial role in human health by facilitating the absorption of dietary lipophilic nutrients, interacting with hormone receptors to regulate host physiology, and shaping gut microbiota composition through antimicrobial activity. Bile acids deconjugation by bacterial bile salt hydrolase has long been recognized as the first necessary bile acid modification required before further transformations can occur. Here, we show that bile salt hydrolase activity is common among human gut bacterial isolates spanning seven major phyla. However, we observed variation in both the extent and the specificity of deconjugation of bile acids among the tested taxa. Unexpectedly, we discovered that certain strains were capable of directly dehydrogenating conjugated bile acids via hydroxysteroid dehydrogenases to produce conjugated secondary bile acids both in vitro and in vivo. These results challenge the prevailing notion that deconjugation is a prerequisite for further bile acid modifications and lay a foundation for new hypotheses regarding how bacteria act individually or in concert to diversify the bile acid pool and influence host physiology.

Intracellular bacteria and protists rely on the host cell to supply many metabolites, but the mechanisms through which pathogens manipulate host metabolism to their benefit are not understood. Here, we demonstrate that when the obligate intracellular parasite Toxoplasma gondii secretes its rhoptry organelle contents into the host cytoplasm before invasion-a process called "kiss and spit"-host cell metabolite abundance is altered in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and amino acid synthesis. U-13C6-labeling metabolomics confirmed that kiss and spit increased the flow of carbon through the pentose phosphate pathway and nucleotide synthesis. An increase in 2,3-bisphosphoglycerate abundance led us to investigate the activation of host cytosolic nucleosidase II (cN-II) to provide purines for the parasite. We found that T. gondii manipulates the host cN-II enzyme to dephosphorylate GMP and IMP that it needs for replication. Furthermore, we found that the approved anti-cancer drug fludarabine, which inhibits cN-II, also inhibits Toxoplasma replication. These results reveal Toxoplasma host cell manipulation and highlight potential therapies for toxoplasmosis.IMPORTANCEA fundamental challenge in parasitology is understanding how intracellular parasites rapidly reprogram host metabolism to support replication. This study reveals that Toxoplasma gondii initiates profound metabolic reprogramming through a "kiss-and-spit" mechanism, secreting effector molecules without invasion. We demonstrate that T. gondii specifically hijacks host cytosolic 5'-nucleotidase II (cN-II) by elevating 2,3-bisphosphoglycerate levels, which allosterically activates this enzyme to generate purines essential for parasite survival. Genetic deletion of host cN-II significantly impairs parasite replication, establishing cN-II as a critical host dependency factor. These findings have important implications for antiparasitic drug development while advancing our understanding of purine metabolism in apicomplexan parasites. More broadly, elucidating the molecular mechanism linking parasite effector secretion to specific host enzyme activation provides a framework for understanding metabolic manipulation across other intracellular pathogens.

Bacterial pathogens must possess finely tuned physiological adaptations to adapt to their infectious niche. One such niche inhabited by Listeria monocytogenes (L. monocytogenes) is the host cell cytosol, a compartment characterized by significant barriers to entry, metabolic limitation, and immune surveillance. Previously, we identified L. monocytogenes transposon mutants defective for intracellular survival due to disruptions in key metabolic pathways, including cell wall biosynthesis, menaquinone production, and pyruvate metabolism. Here, we demonstrate that mutations in the pyruvate dehydrogenase (PDH) complex exhibit pronounced survival defects during infection, despite retaining robust growth and survival in nutrient-rich media. Metabolomic profiling of the PDH E2 subunit mutant revealed an altered respiro-fermentative metabolism with lower levels of both upper glycolytic intermediates and tricarboxylic acid cycle intermediates coupled with elevated levels of pyruvate and lactate. Additionally, we found that PDH mutants are unable to efficiently utilize phosphotransferase system (PTS)-dependent carbon sources, but their growth is indistinguishable from that of the wild type on non-PTS carbon sources such as hexose phosphates. A suppressor screen identified five suppressor mutants with restored ability to grow on the PTS substrate fructose, and each contained an independent mutation in the redox-sensing regulator rex. Loss of Rex function in PDH mutants partially restored intracellular growth, but not virulence in vivo. Together, these findings demonstrate that PDH is required for the import and metabolism of PTS-dependent carbon sources in the host cytosol and suggest that PDH-dependent redox balance and respiro-fermentative metabolism ultimately contribute to intracellular fitness and virulence.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately one-third of the global population and can progress to metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis, increasing the risk of cirrhosis, hepatocellular carcinoma, and mortality. Gut microbes driven by diets high in saturated fat, simple sugar, and cholesterol contribute to disease progression, yet the underlying mechanisms remain undefined. We explored the independent and synergistic effects of dietary saturated fat and cholesterol on MASH development using specific pathogen-free (SPF) and germ-free (GF) mice. We demonstrate that (1) both dietary cholesterol and saturated fat are required to induce fibrosing MASH in SPF mice, whereas GF mice are protected, (2) saturated fat and cholesterol individually alter gut microbial membership, potentially via altered bile acid metabolism, while their combination promotes a distinct composition, including an increase in Parasutterella spp. which correlates with hepatic fibrosis, and (3) diluted cecal contents from SPF, but not GF, mice fed high-fat, high-cholesterol diets are enriched in deoxycholic acid and activate human hepatic stellate cells in vitro, suggesting a mechanistic link between dietary lipid-induced microbiota and liver fibrogenesis. These findings reveal how specific Western dietary components shape the gut microbiota and contribute to hepatic fibrosis via stellate cell activation, offering potential targets for therapeutic interventions against MASLD/MASH.

Identifying design principles for robust inhibition of human pathogens is a major goal of microbiome engineering. By building synthetic microbial communities from the bottom-up guided by Bayesian active learning, we investigate the Clostridioides difficile growth landscape across thousands of species-metabolite conditions. Mechanistic consumer resource and machine learning models uncover significant interactions linking metabolites and species, and exhibit concordance with microbial interactions identified in human microbiome datasets. Guided by machine learning and mechanistic models, we elucidate microbial communities capable of robustly inhibiting C. difficile across diverse nutrient environments in vitro. Metabolomic profiling identified sorbitol, mannitol, and proline as key metabolites mediating community-driven C. difficile inhibition. Model-optimized communities significantly reduced C. difficile colonization in the murine gut whereas a model-designed non-inhibitory community did not exhibit this property. Communities display consistent metabolomic patterns in vitro and in vivo, suggesting that resource competition is a design principle of robust inhibition. Together, these findings establish an integrated experimental and computational framework for the rational design of microbial communities that robustly suppress pathogens across diverse environmental contexts.

Identifying metabolites and metabolic reactions specific to a cellular state, such as inflammatory state in immune cells, is of great interest, as it can provide important biomarkers and point to compounds and reactions of specific biological functions. However, many cell state-specific metabolites remain in the unannotated part of metabolome. Here we identified a series of sulfur-containing metabolites that are actively produced in macrophages upon classical activation, but not in resting state or alternative activation state. Isotopic tracing, in vitro assays and genetic perturbations further revealed that they are formed from reactions between free cysteine and several important intermediates in glycolysis and TCA cycle. Upon classical activation, macrophages specifically upregulate the import of cystine via Slc7a11, supporting the production of these adducts. Their production dynamically responds to changes in central metabolism, environmental nutrient levels, and is regulated by nitric oxide. Finally, we confirmed these newly identified compounds also present in human samples, and most of them are significantly elevated in inflammatory granuloma annulare lesions. This work elucidated a previously uncharted part of metabolic network that is associated with inflammation and metabolic stress condition, which has important implications and set foundation for many future discoveries.

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species—Zymomonas mobilis, Escherichia coli, and Clostridium thermocellum—which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding in vivo metabolic fluxes and ΔG measurements, we found that the highly favorable Entner-Doudoroff pathway in Z. mobilis requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in C. thermocellum, with the Embden-Meyerhof-Parnas pathway in E. coli exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in C. thermocellum requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in Z. mobilis. Thus, protein investment across glycolytic pathways reflects differences in their thermodynamic favorability.IMPORTANCECells regulate metabolic fluxes to balance energy production, biosynthesis, and the efficient use of limited resources, including the finite capacity for synthesizing and maintaining metabolic enzymes. Here, we present in vivo evidence that strongly thermodynamically favorable metabolic pathways require significantly fewer enzyme resources to sustain a given flux compared to less thermodynamically favorable pathways. These findings underscore the connection between pathway thermodynamics, resource allocation, and enzyme burden, providing valuable insights for metabolic engineering strategies aimed at optimizing pathways for high flux with minimal protein cost.

ABSTRACT Guanosine triphosphate (GTP) is essential for macromolecular biosynthesis, and its intracellular levels are tightly regulated in bacteria. Loss of the alarmone (p)ppGpp disrupts GTP regulation in Bacillus subtilis, causing cell death in the presence of exogenous guanosine and underscoring the critical importance of GTP homeostasis. To investigate the basis of guanosine toxicity, we performed a genetic selection for spontaneous mutations that suppress this effect, uncovering an unexpected link between GTP synthesis and glycolysis. In particular, we identified suppressor mutations in pyk, which encodes pyruvate kinase, a glycolytic enzyme. Metabolomic analysis revealed that inactivating pyruvate kinase prevents guanosine toxicity by reducing GTP levels. Although traditionally associated with ATP generation via substrate-level phosphorylation, B. subtilis pyruvate kinase in vitro was found to produce GTP and UTP approximately 10 and three times more efficiently than ATP, respectively. This efficient GTP/UTP synthesis extends to Enterococcus faecalis and Listeria monocytogenes, challenging the conventional understanding of pyruvate kinase’s primary role in ATP production. These findings support a model in which glycolysis directly contributes to GTP synthesis, fueling energy-demanding processes, such as protein translation. Finally, we observed a synergistic essentiality of the Δndk Δpyk double mutant specifically on glucose, indicating that pyruvate kinase and nucleoside diphosphate kinase are the major contributors to nucleoside triphosphate production and complement each other during glycolysis. Our work highlights the critical role of nucleotide selectivity in pyruvate kinase and its broader implications in cellular physiology.IMPORTANCEIn this study, we reveal that pyruvate kinase, a key glycolytic enzyme, primarily generates GTP from GDP in Bacillus subtilis, relative to other nucleotide triphosphates, such as ATP. This finding, uncovered through genetic selection for mutants that suppress toxic GTP overaccumulation, challenges the conventional understanding that pyruvate kinase predominantly produces ATP via substrate-level phosphorylation. The substantial role of GTP production by pyruvate kinase suggests a model where glycolysis rapidly and directly supplies GTP as the energy currency to power high GTP-demanding processes such as protein synthesis. Our results underscore the importance of nucleotide selectivity (ATP vs GTP vs UTP) in shaping the physiological state and fate of the cell, prompting further exploration into the mechanisms and broader implications of this selective nucleotide synthesis.

The evolutionarily conserved GATA-type transcription factor (TF) NsdD regulates sexual and asexual development as well as secondary metabolism in various Aspergillus species. Despite its well-known multifunctionality, the mechanisms by which NsdD coordinates such diverse biological processes remain unclear. To address this gap, we have conducted network-based multiomics analyses in two distantly related species, Aspergillus nidulans and Aspergillus flavus. Transcriptomic profiling reveals that NsdD regulates gene expression in a cell type- and species-specific manner. The potential evolutionary conservation of NsdD was tested by a cross-complementation experiment in which the A. nidulans nsdD gene was introduced into the A. flavus ΔnsdD mutant. This partially restored key phenotypes and gene expression profiles but failed to fully recapitulate wild-type regulation, suggesting species-specific functionality. To further dissect NsdD's roles, we have performed genome-wide ChIP-seq analyses and identified 502 and 674 potential direct targets in A. nidulans and A. flavus, respectively, including major developmental and metabolic regulators such as veA, flbD, brlA, vosA, rosA, and laeA. Motif analysis reveals a conserved NsdD binding site (5'-GATCT-3'), designated as the NsdD response element. Network analyses uncover core regulatory modules and reveal extensive gene regulatory network (GRN) rewiring between the two species. While NsdD governs conserved biological processes, divergence in its direct targets and downstream interactions contributes to species-specific traits, including differences in asexual morphology and production of sterigmatocystin/aflatoxin. This study provides the first genome-wide comparative map of NsdD-mediated GRNs in filamentous fungi and highlights how evolutionary rewiring allows a conserved TF to acquire distinct regulatory functions across species.IMPORTANCEMultifunctional TFs are central in coordinating development and metabolism in filamentous fungi. In this study, we systematically dissect the regulatory functions of NsdD, a highly conserved GATA-type TF in Pezizomycotina, using network-based multi-omics approaches in two distantly related species, A. nidulans and A. flavus. Our analyses reveal that NsdD governs fungal development and metabolism through species-specific GRNs, directly targeting key upstream regulators and genes involved in core cellular processes. These regulatory distinctions underlie the morphological and metabolic differences observed between the two species. Notably, our cross-species comparison uncovers extensive GRN rewiring, demonstrating how evolutionary divergence can reshape transcriptional networks even under conserved regulatory control. The resulting GRN maps offer a valuable framework for understanding gene regulation in Aspergillus and provide a foundation for broader studies on the evolution of transcriptional networks and conserved regulatory factors in filamentous fungi.

Intracellular pools of deoxynucleoside triphosphates (dNTPs) are strictly maintained throughout the cell cycle to ensure accurate and efficient DNA replication. DNA synthesis requires an abundance of dNTPs, but elevated dNTP concentrations in nonreplicating cells delay entry into S phase. Enzymes known as deoxyguanosine triphosphate triphosphohydrolases (Dgts) hydrolyze dNTPs into deoxynucleosides and triphosphates, and we propose that Dgts restrict dNTP concentrations to promote the G1 to S phase transition. We characterized a Dgt from the bacterium Caulobacter crescentus termed flagellar signaling suppressor C (fssC) to clarify the role of Dgts in cell cycle regulation. Deleting fssC increases dNTP levels and extends the G1 phase of the cell cycle through a mechanism independent of the response regulator CtrA. Segregation and duplication of the chromosomal origin of replication (oriC) are delayed in ∆fssC, but the rate of replication elongation is unchanged. We conclude that dNTP hydrolysis by FssC promotes the initiation of DNA replication. This work further establishes Dgts as important regulators of the G1 to S phase transition, and the high conservation of Dgts across all domains of life implies that Dgt-dependent cell cycle control may be widespread in many organisms.IMPORTANCECells must faithfully replicate their genetic material in order to proliferate. Studying the regulatory pathways that determine when a cell initiates DNA replication is important for understanding fundamental biological processes, and it can also improve the strategies used to treat diseases that affect the cell cycle. Here, we identify a nucleotide signaling pathway that influences when cells begin DNA replication. We show that this pathway promotes the transition from the G1 to the S phase of the cell cycle in the bacterium Caulobacter crescentus and propose that this pathway is prevalent in all domains of life.

Hypercholesterolemia contributes to the development of atherosclerosis and is a major risk factor for cardiovascular diseases (CVD). Dietary fiber can attenuate CVD, at least in part, by serving as a fermentable substrate for gut bacteria, leading to the production of short-chain fatty acids (SCFAs), such as butyrate and propionate, which have been linked to atheroprotective effects. SCFAs are sensed by G-protein coupled receptors including GPR41, GPR43, and GPR109A. To explore the role of these receptors in hypercholesterolemia and CVD, we examined atherosclerosis progression and lipid metabolism in Gpr41-/-, Gpr43-/-, and Gpr109a-/- mice using a proprotein convertase subtilisin/kexin type 9 adeno-associated virus (PCSK9-AAV) model of hypercholesterolemia. Deficiency of any single SCFA receptor did not significantly affect atherosclerotic plaque burden compared with wild-type (WT) littermates. However, male Gpr41-/- mice exhibited decreased gonadal fat, plasma triacylglycerol, and low-density lipoprotein cholesterol levels compared to their WT littermates. GPR41 deficiency in males was also associated with increased cecal propionate levels, reduced ileal expression of nutrient transporters such as Npc1l1 and a trend toward increased gut motility. In addition, male Gpr41-/- mice displayed altered gut microbiota composition and lower levels of microbially generated bile acids relative to their WT counterparts. Together, these findings highlight GPR41 as a key intestinal chemosensor regulating nutrient uptake, lipid storage, and microbiota composition.

Microbial strains engineered for high-titer ethanol production remain limited in achievable titers compared to native producers such as Zymomonas mobilis. A central unresolved question is why fermentation ceases in such engineered strains before maximum titers are reached. Here, we integrate metabolite profiling with thermodynamic analysis to examine this phenomenon in engineered Escherichia coli and Thermoanaerobacterium saccharolyticum compared with Z. mobilis. In the engineered strains, fermentation cessation coincided with marked pyruvate accumulation. Max-Min Driving Force (MDF) analysis indicated that the pyruvate kinase step in glycolysis approaches thermodynamic equilibrium under physiological metabolite concentrations, reducing the driving force for the pyruvate-to-ethanol branch. Relaxing constraints on pyruvate and related metabolites restored positive MDF values, implicating thermodynamic limitations as the underlying constraint. By contrast, Z. mobilis maintained positive MDF scores without metabolite accumulation, underscoring an organism-specific strategy that avoids this limitation. These findings establish a systems-level framework linking metabolite concentrations to pathway thermodynamics and highlight opportunities for improving microbial performance in ethanol and other bioproduction contexts.

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC-MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner-Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.IMPORTANCEBiomass pretreatment processes release fermentable sugars from lignocellulosic biomass, but they also generate inhibitors that can impact microbial metabolism. This study provides a systems-level evaluation of how Zymomonas mobilis responds to hydrolysate stress, revealing distinct physiological and lipid membrane remodeling responses. While some stress responses overlap with those induced by ethanol and isobutanol toxicity, both valuable biofuels, hydrolysate exposure elicits unique metabolic shifts. These findings offer valuable insights for engineering Z. mobilis strains with improved tolerance and performance for efficient bioconversion of lignocellulosic hydrolysates into biofuels and bioproducts.

Microbes in the intestine transform bile acids during transit, altering their functional and signaling capacities before recirculation via the portal vein. Sex differences in the gut microbiota have been noted, but their consequence on bile acid composition is unclear. Here, we investigated the composition and functional potential of microbes in the small and large intestines together with portal and systemic bile acid levels. Female and male mice exhibit distinct microbial diversity throughout the length of the intestine, leading to dimorphism in genes related to bile acid transformation. Of note, genes linked to bacterial oxidative properties were abundant in males, and consistently, we found 3X higher oxo-bile acids in portal circulation in males than females. Conversely, conjugated primary bile acids were 1.8X more abundant in the portal bile acid pool of female mice. Oxidized and deconjugated bile acids were absent in germ-free mice consistent with microbe-mediated bile acid transformation. More importantly, gnotobiotic mice do not show sex differences in portal bile acids. Taken together, we demonstrate that sex differences in gut microbiota with subsequent changes in microbially transformed bile acid levels contribute to distinct sex-specific bile acid pools within the enterohepatic loop.