Cuproptosis in cancer: biological implications and therapeutic opportunities.

Cuproptosis, a newly identified copper (Cu)-dependent form of cell death, stands out due to its distinct mechanism that sets it apart from other known cell death pathways. The molecular underpinnings of cuproptosis involve the binding of Cu to lipoylated enzymes in the tricarboxylic acid cycle. This interaction triggers enzyme aggregation and proteotoxic stress, culminating in cell death. The specific mechanism of cuproptosis has yet to be fully elucidated. This newly recognized form of cell death has sparked numerous investigations into its role in tumorigenesis and cancer therapy. In this review, we summarized the current knowledge on Cu metabolism and its link to cancer. Furthermore, we delineated the molecular mechanisms of cuproptosis and summarized the roles of cuproptosis-related genes in cancer. Finally, we offered a comprehensive discussion of the most recent advancements in Cu ionophores and nanoparticle delivery systems that utilize cuproptosis as a cutting-edge strategy for cancer treatment.

Lactate transported by MCT1 plays an active role in promoting mitochondrial biogenesis and enhancing TCA flux in skeletal muscle.

Once considered as a "metabolic waste," lactate is now recognized as a major fuel for tricarboxylic acid (TCA) cycle. Our metabolic flux analysis reveals that skeletal muscle mainly uses lactate to fuel TCA cycle. Lactate is transported through the cell membrane via monocarboxylate transporters (MCTs) in which MCT1 is highly expressed in the muscle. We analyzed how MCT1 affects muscle functions using mice with specific deletion of MCT1 in skeletal muscle. MCT1 deletion enhances running performance, increases oxidative fibers while decreasing glycolytic fibers, and enhances flux of glucose to TCA cycle. MCT1 deficiency increases the expression of mitochondrial proteins, augments cell respiration rate, and elevates mitochondrial activity in the muscle. Mechanistically, the protein level of PGC-1α, a master regulator of mitochondrial biogenesis, is elevated upon loss of MCT1 via increases in cellular NAD+ level and SIRT1 activity. Collectively, these results demonstrate that MCT1-mediated lactate shuttle plays a key role in regulating muscle functions by modulating mitochondrial biogenesis and TCA flux.

Succinate promotes pulmonary fibrosis through GPR91 and predicts death in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is believed to be associated with a notable disruption of cellular energy metabolism. By detecting the changes of energy metabolites in the serum of patients with pulmonary fibrosis, we aimed to investigate the diagnostic and prognostic value of energy metabolites in IPF, and further elucidated the mechanism of their involvement in pulmonary fibrosis. Through metabolomics research, it was discovered that the TCA cycle intermediates changed dramatically in IPF patients. In another validation cohort of 55 patients with IPF compared to 19 healthy controls, it was found that succinate, an intermediate product of TCA cycle, has diagnostic and prognostic value in IPF. The cut-off levels of serum succinate were 98.36 µM for distinguishing IPF from healthy controls (sensitivity, 83.64%; specificity, 63.16%; likelihood ratio, 2.27, respectively). Moreover, a high serum succinate level was independently associated with higher rates of disease progression (OR 13.087, 95%CI (2.819-60.761)) and mortality (HR 3.418, 95% CI (1.308-8.927)). In addition, accumulation of succinate and increased expression of the succinate receptor GPR91 were found in both IPF patients and BLM mouse models of pulmonary fibrosis. Reducing succinate accumulation in BLM mice alleviated pulmonary fibrosis and 21d mortality, while exogenous administration of succinate can aggravate pulmonary fibrosis in BLM mice. Furthermore, GPR91 deficiency protected against lung fibrosis caused by BLM. In vitro, succinate promoted the activation of lung fibroblasts by activating ERK pathway through GPR91. In summary, succinate is a promising biomarker for diagnosis and prognosis of IPF. The accumulation of succinate may promote fibroblast activation through GPR91 and pulmonary fibrosis.

Metabolic reprogramming-driven homologous recombination and TCA cycle dysregulation contribute to poor prognoses in lung adenocarcinoma.

Increasing evidence has shown that homologous recombination (HR) and metabolic reprogramming are essential for cellular homeostasis. These two processes are independent as well as closely intertwined. Nevertheless, they have rarely been reported in lung adenocarcinoma (LUAD). We analysed the genomic, immune microenvironment and metabolic microenvironment features under different HR activity states. Using cell cycle, EDU and cell invasion assays, we determined the impacts of si-SHFM1 on the LUAD cell cycle, proliferation and invasion. The levels of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) were determined by ELISA in the NC and si-SHFM1 groups of A549 cells. Finally, cell samples were used to extract metabolites for HPIC-MS/MS to analyse central carbon metabolism. We found that high HR activity was associated with a poor prognosis in LUAD, and HR was an independent prognostic factor for TCGA-LUAD patients. Moreover, LUAD samples with a high HR activity presented low immune infiltration levels, a high degree of genomic instability, a good response status to immune checkpoint blockade therapy and a high degree of drug sensitivity. The si-SHFM1 group presented a significantly higher proportion of cells in the G0/G1 phase, lower levels of DNA replication, and significantly lower levels of cell migration and both TCA enzymes. Our current results indicated that there is a strong correlation between HR and the TCA cycle in LUAD. The TCA cycle can promote SHFM1-mediated HR in LUAD, raising their activities, which can finally result in a poor prognosis and impair immunotherapeutic efficacy.

Co-expression analysis reveals distinct alliances around two carbon fixation pathways in hydrothermal vent symbionts.

Most autotrophic organisms possess a single carbon fixation pathway. The chemoautotrophic symbionts of the hydrothermal vent tubeworm Riftia pachyptila, however, possess two functional pathways: the Calvin-Benson-Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. How these two pathways are coordinated is unknown. Here we measured net carbon fixation rates, transcriptional/metabolic responses and transcriptional co-expression patterns of Riftia pachyptila endosymbionts by incubating tubeworms collected from the East Pacific Rise at environmental pressures, temperature and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes; the rTCA is allied to hydrogenases and dissimilatory nitrate reduction, whereas the CBB is allied to sulfide oxidation and assimilatory nitrate reduction, suggesting distinctive yet complementary roles in metabolic function. Furthermore, our network analysis implicates the rTCA and a group 1e hydrogenase as key players in the physiological response to limitation of sulfide and oxygen. Net carbon fixation rates were also exemplary, and accordingly, we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.

Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism.

Enzymes of the central metabolism tend to assemble into transient supramolecular complexes. However, the functional significance of the interactions, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from Bacillus subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in phase separated droplets we show that MDH-ICD interaction leads to enzyme agglomeration with a concomitant enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory demonstrates that MDH-mediated clustering of ICD molecules explains the observed phenomena. In vivo analyses reveal that MDH overexpression leads to accumulation of 2-oxoglutarate and reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings suggest that the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism.

The blood serum metabolome profile after different phases of a 4-km cycling time trial: Secondary analysis of a randomized controlled trial.

It has been assumed that exercise intensity variation throughout a cycling time trial (TT) occurs in alignment of various metabolic changes to prevent premature task failure. However, this assumption is based on target metabolite responses, which limits our understanding of the complex interconnection of metabolic responses during exercise. The current study characterized the metabolomic profile, an untargeted metabolic analysis, after specific phases of a cycling 4-km TT. Eleven male cyclists performed three separated TTs in a crossover counterbalanced design, which were interrupted at the end of the fast-start (FS, 600 ± 205 m), even-pace (EP, 3600 ± 190 m), or end-spurt (ES, 4000 m) phases. Blood samples were taken before any exercise and 5 min after exercise cessation, and the metabolomic profile characterization was performed using Nuclear Magnetic Resonance metabolomics. Power output (PO) was also continually recorded. There were higher PO values during the FS and ES compared to the EP (all p < 0.05), which were accompanied by distinct metabolomic profiles. FS showed high metabolite expression in TCA cycle and its related pathways (e.g., glutamate, citric acid, and valine metabolism); whereas, the EP elicited changes associated with antioxidant effects and oxygen delivery adjustment. Finally, ES was related to pathways involved in NAD turnover and serotonin metabolism. These findings suggest that the specific phases of a cycling TT are accompanied by distinct metabolomic profiles, providing novel insights regarding the relevance of specific metabolic pathways on the process of exercise intensity regulation.

Cellular succinate metabolism and signaling in inflammation: implications for therapeutic intervention.

Succinate, traditionally viewed as a mere intermediate of the tricarboxylic acid (TCA) cycle, has emerged as a critical mediator in inflammation. Disruptions within the TCA cycle lead to an accumulation of succinate in the mitochondrial matrix. This excess succinate subsequently diffuses into the cytosol and is released into the extracellular space. Elevated cytosolic succinate levels stabilize hypoxia-inducible factor-1α by inhibiting prolyl hydroxylases, which enhances inflammatory responses. Notably, succinate also acts extracellularly as a signaling molecule by engaging succinate receptor 1 on immune cells, thus modulating their pro-inflammatory or anti-inflammatory activities. Alterations in succinate levels have been associated with various inflammatory disorders, including rheumatoid arthritis, inflammatory bowel disease, obesity, and atherosclerosis. These associations are primarily due to exaggerated immune cell responses. Given its central role in inflammation, targeting succinate pathways offers promising therapeutic avenues for these diseases. This paper provides an extensive review of succinate's involvement in inflammatory processes and highlights potential targets for future research and therapeutic possibilities development.

Bidirectional modulation of TCA cycle metabolites and anaplerosis by metformin and its combination with SGLT2i.

BACKGROUND: Metformin and sodium-glucose-cotransporter-2 inhibitors (SGLT2i) are cornerstone therapies for managing hyperglycemia in diabetes. However, their detailed impacts on metabolic processes, particularly within the citric acid (TCA) cycle and its anaplerotic pathways, remain unclear. This study investigates the tissue-specific metabolic effects of metformin, both as a monotherapy and in combination with SGLT2i, on the TCA cycle and associated anaplerotic reactions in both mice and humans. METHODS: Metformin-specific metabolic changes were initially identified by comparing metformin-treated diabetic mice (MET) with vehicle-treated db/db mice (VG). These findings were then assessed in two human cohorts (KORA and QBB) and a longitudinal KORA study of metformin-naïve patients with Type 2 Diabetes (T2D). We also compared MET with db/db mice on combination therapy (SGLT2i + MET). Metabolic profiling analyzed 716 metabolites from plasma, liver, and kidney tissues post-treatment, using linear regression and Bonferroni correction for statistical analysis, complemented by pathway analyses to explore the pathophysiological implications. RESULTS: Metformin monotherapy significantly upregulated TCA cycle intermediates such as malate, fumarate, and α-ketoglutarate (α-KG) in plasma, and anaplerotic substrates including hepatic glutamate and renal 2-hydroxyglutarate (2-HG) in diabetic mice. Downregulated hepatic taurine was also observed. The addition of SGLT2i, however, reversed these effects, such as downregulating circulating malate and α-KG, and hepatic glutamate and renal 2-HG, but upregulated hepatic taurine. In human T2D patients on metformin therapy, significant systemic alterations in metabolites were observed, including increased malate but decreased citrulline. The bidirectional modulation of TCA cycle intermediates in mice influenced key anaplerotic pathways linked to glutaminolysis, tumorigenesis, immune regulation, and antioxidative responses. CONCLUSION: This study elucidates the specific metabolic consequences of metformin and SGLT2i on the TCA cycle, reflecting potential impacts on the immune system. Metformin shows promise for its anti-inflammatory properties, while the addition of SGLT2i may provide liver protection in conditions like metabolic dysfunction-associated steatotic liver disease (MASLD). These observations underscore the importance of personalized treatment strategies.

Reprogrammed mitochondria: a central hub of cancer cell metabolism.

Mitochondria represent the metabolic hub of normal cells and play this role also in cancer but with different functional purposes. While cells in differentiated tissues have the prerogative of maintaining basal metabolism and support the biosynthesis of specialized products, cancer cells have to rewire the metabolic constraints imposed by the differentiation process. They need to balance the bioenergetic supply with the anabolic requirements that entail the intense proliferation rate, including nucleotide and membrane lipid biosynthesis. For this aim, mitochondrial metabolism is reprogrammed following the activation of specific oncogenic pathways or due to specific mutations of mitochondrial proteins. The main process leading to mitochondrial metabolic rewiring is the alteration of the tricarboxylic acid cycle favoring the appropriate orchestration of anaplerotic and cataplerotic reactions. According to the tumor type or the microenvironmental conditions, mitochondria may decouple glucose catabolism from mitochondrial oxidation in favor of glutaminolysis or disable oxidative phosphorylation for avoiding harmful production of free radicals. These and other metabolic settings can be also determined by the neo-production of oncometabolites that are not specific for the tissue of origin or the accumulation of metabolic intermediates able to boost pro-proliferative metabolism also impacting epigenetic/transcriptional programs. The full characterization of tumor-specific mitochondrial signatures may provide the identification of new biomarkers and therapeutic opportunities based on metabolic approaches.

Deficits in brain glucose transport among younger adults with obesity.

OBJECTIVE: Obesity is associated with alterations in eating behavior and neurocognitive function. In this study, we investigate the effect of obesity on brain energy utilization, including brain glucose transport and metabolism. METHODS: A total of 11 lean participants and 7 young healthy participants with obesity (mean age, 27 years) underwent magnetic resonance spectroscopy scanning coupled with a hyperglycemic clamp (target, ~180 mg/dL) using [1-13C] glucose to measure brain glucose uptake and metabolism, as well as peripheral markers of insulin resistance. RESULTS: Individuals with obesity demonstrated an ~20% lower ratio of brain glucose uptake to cerebral glucose metabolic rate (Tmax/CMRglucose) than lean participants (2.12 ± 0.51 vs. 2.67 ± 0.51; p = 0.04). The cerebral tricarboxylic acid cycle flux (VTCA) was similar between the two groups (p = 0.64). There was a negative correlation between total nonesterified fatty acids and Tmax/CMRglucose (r = -0.477; p = 0.045). CONCLUSIONS: We conclude that CMRglucose is unlikely to differ between groups due to similar VTCA, and, therefore, the glucose transport Tmax is lower in individuals with obesity. These human findings suggest that obesity is associated with reduced cerebral glucose transport capacity even at a young age and in the absence of other cardiometabolic comorbidities, which may have implications for long-term brain function and health.

Mechanisms associated with cuproptosis and implications for ovarian cancer.

Ovarian cancer, a profoundly fatal gynecologic neoplasm, exerts a substantial economic strain on nations globally. The formidable challenge of its frequent relapse necessitates the exploration of novel cytotoxic agents, efficacious antineoplastic medications with minimal adverse effects, and strategies to surmount resistance to primary chemotherapeutic agents. These endeavors aim to supplement extant pharmacological interventions and elucidate molecular mechanisms underlying induced cytotoxicity, distinct from conventional therapeutic modalities. Recent scientific research has unveiled a novel form of cellular demise, known as copper-death, which is contingent upon the intracellular concentration of copper. Diverging from conventional mechanisms of cellular demise, copper-death exhibits a pronounced reliance on mitochondrial respiration, particularly the tricarboxylic acid (TCA) cycle. Tumor cells manifest distinctive metabolic profiles and elevated copper levels in comparison to their normal counterparts. The advent of copper-death presents alluring possibilities for targeted therapeutic interventions within the realm of cancer treatment. Hence, the primary objective of this review is to present an overview of the proteins and intricate mechanisms associated with copper-induced cell death, while providing a comprehensive summary of the knowledge acquired regarding potential therapeutic approaches for ovarian cancer. These findings will serve as valuable references to facilitate the advancement of customized therapeutic interventions for ovarian cancer.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Itaconate reduces proliferation and migration of fibroblast-like synoviocytes and ameliorates arthritis models.

Fibroblast-like synoviocytes (FLS) play critical roles in rheumatoid arthritis (RA). Itaconate (ITA), an endogenous metabolite derived from the tricarboxylic acid (TCA) cycle, has attracted attention because of its anti-inflammatory, antiviral, and antimicrobial effects. This study evaluated the effect of ITA on FLS and its potential to treat RA. ITA significantly decreased FLS proliferation and migration in vitro, as well as mitochondrial oxidative phosphorylation and glycolysis measured by an extracellular flux analyzer. ITA accumulates metabolites including succinate and citrate in the TCA cycle. In rats with type II collagen-induced arthritis (CIA), intra-articular injection of ITA reduced arthritis and bone erosion. Irg1-deficient mice lacking the ability to produce ITA had more severe arthritis than control mice in the collagen antibody-induced arthritis. ITA ameliorated CIA by inhibiting FLS proliferation and migration. Thus, ITA may be a novel therapeutic agent for RA.

Quercetin induces itaconic acid-mediated M1/M2 alveolar macrophages polarization in respiratory syncytial virus infection.

BACKGROUND: Quercetin has received extensive attention for its therapeutic potential treating respiratory syncytial virus (RSV) infection diseases. Recent studies have highlighted quercetin's ability of suppressing alveolar macrophages (AMs)-derived lung inflammation. However, the anti-inflammatory mechanism of quercetin against RSV infection still remains elusive. PURPOSE: This study aims to elucidate the mechanism about quercetin anti-inflammatory effect on RSV infection. METHODS: BALB/c mice were intranasally infected with RSV and received quercetin (30, 60, 120 mg/kg/d) orally for 3 days. Additionally, an in vitro infection model utilizing mouse alveolar macrophages (MH-S cells) was employed to validate the proposed mechanism. RESULTS: Quercetin exhibited a downregulatory effect on glycolysis and tricarboxylic acid (TCA) cycle metabolism in RSV-infected AMs. However, it increased itaconic acid production, a metabolite derived from citrate through activating immune responsive gene 1 (IRG1), and further inhibiting succinate dehydrogenase (SDH) activity. While the suppression of SDH activity orchestrated a cascading downregulation of Hif-1α/NLRP3 signaling, ultimately causing AMs polarization from M1 to M2 phenotypes. CONCLUSION: Our study demonstrated quercetin stimulated IRG1-mediated itaconic acid anabolism and further inhibited SDH/Hif-1α/NLRP3 signaling pathway, which led to M1 to M2 polarization of AMs so as to ameliorate RSV-induced lung inflammation.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

Exploring the mystery of tumor metabolism: Warburg effect and mitochondrial metabolism fighting side by side.

The metabolic reconfiguration of tumor cells constitutes a pivotal aspect of tumor proliferation and advancement. This study delves into two primary facets of tumor metabolism: the Warburg effect and mitochondrial metabolism, elucidating their contributions to tumor dominance. The Warburg effect facilitates efficient energy acquisition by tumor cells through aerobic glycolysis and lactic acid fermentation, offering metabolic advantages conducive to growth and proliferation. Simultaneously, mitochondrial metabolism, serving as the linchpin of sustained tumor vitality, orchestrates the tricarboxylic acid cycle and electron transport chain, furnishing a steadfast and dependable wellspring of biosynthesis for tumor cells. Regarding targeted therapy, this discourse examines extant strategies targeting tumor glycolysis and mitochondrial metabolism, underscoring their potential efficacy in modulating tumor metabolism while envisaging future research trajectories and treatment paradigms in the realm of tumor metabolism. By means of a thorough exploration of tumor metabolism, this study aspires to furnish crucial insights into the regulation of tumor metabolic processes, thereby furnishing valuable guidance for the development of novel therapeutic modalities. This comprehensive deliberation is poised to catalyze advancements in tumor metabolism research and offer novel perspectives and pathways for the formulation of cancer treatment strategies in the times ahead.

The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.