SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway.

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.

Metabolic reprogramming driven by EZH2 inhibition depends on cell-matrix interactions.

EZH2 (Enhancer of Zeste Homolog 2), a subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), which represses expression of genes. It also has PRC2-independent functions, including transcriptional coactivation of oncogenes, and is frequently overexpressed in lung cancers. Clinically, EZH2 inhibition can be achieved with the FDA-approved drug EPZ-6438 (tazemetostat). To realize the full potential of EZH2 blockade, it is critical to understand how cell-cell/cell-matrix interactions present in 3D tissue and cell culture systems influences this blockade in terms of growth-related metabolic functions. Here, we show that EZH2 suppression reduced growth of human lung adenocarcinoma A549 cells in 2D cultures but stimulated growth in 3D cultures. To understand the metabolic underpinnings, we employed [13C6]-glucose stable isotope-resolved metabolomics to determine the effect of EZH2 suppression on metabolic networks in 2D versus 3D A549 cultures. The Krebs cycle, neoribogenesis, γ-aminobutyrate metabolism, and salvage synthesis of purine nucleotides were activated by EZH2 suppression in 3D spheroids but not in 2D cells, consistent with the growth effect. Using simultaneous 2H7-glucose + 13C5,15N2-Gln tracers and EPZ-6438 inhibition of H3 trimethylation, we delineated the effects on the Krebs cycle, γ-aminobutyrate metabolism, gluconeogenesis, and purine salvage to be PRC2-dependent. Furthermore, the growth/metabolic effects differed for mouse Matrigel versus self-produced A549 extracellular matrix. Thus, our findings highlight the importance of the presence and nature of extracellular matrix in studying the function of EZH2 and its inhibitors in cancer cells for modeling the in vivo outcomes.

Viral AMGs-driven pentose phosphate pathway in natural wetland.

Viruses exist anywhere on earth where there is life, and among them, virus-encoded auxiliary metabolic genes (AMGs) can maintain ecosystem balance and play a major role in the global ecosystem. Although the function of AMGs has been widely reported, the genetic diversity of AMGs in natural ecosystems is still poorly understood. Exploring the genetic diversity of viral community-wide AMGs is essential to gain insight into the complex interactions between viruses and hosts. In this article, we studied the phylogenetic tree, principal co-ordinates analysis (PCoA), α diversity, and metabolic pathways of viral auxiliary metabolism genes involved in the pentose phosphate pathway (PPP) through metagenomics, and the changes of metabolites and genes of host bacteria were further studied by using Pseudomonas mandelii SW-3 and its lytic phage based on metabolic flow and AMGs expression. We found that the viral AMGs in the Napahai plateau wetland were created by a combination of various external forces, which contributed to the rich genetic diversity, uniqueness, and differences of the virus, which promoted the reproduction of offspring and better adaptation to the environment. Overall, this study systematically describes the genetic diversity of AMGs associated with the PPP in plateau wetland ecosystems and further expands the understanding of phage-host unique interactions.

PBX3 promotes pentose phosphate pathway and colorectal cancer progression by enhancing G6PD expression.

Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted G6PD transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential in vitro and in vivo. Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.

Streamlined identification of strain engineering targets for bioprocess improvement using metabolic pathway enrichment analysis.

Metabolomics is a powerful tool for the identification of genetic targets for bioprocess optimisation. However, in most cases, only the biosynthetic pathway directed to product formation is analysed, limiting the identification of these targets. Some studies have used untargeted metabolomics, allowing a more unbiased approach, but data interpretation using multivariate analysis is usually not straightforward and requires time and effort. Here we show, for the first time, the application of metabolic pathway enrichment analysis using untargeted and targeted metabolomics data to identify genetic targets for bioprocess improvement in a more streamlined way. The analysis of an Escherichia coli succinate production bioprocess with this methodology revealed three significantly modulated pathways during the product formation phase: the pentose phosphate pathway, pantothenate and CoA biosynthesis and ascorbate and aldarate metabolism. From these, the two former pathways are consistent with previous efforts to improve succinate production in Escherichia coli. Furthermore, to the best of our knowledge, ascorbate and aldarate metabolism is a newly identified target that has so far never been explored for improving succinate production in this microorganism. This methodology therefore represents a powerful tool for the streamlined identification of strain engineering targets that can accelerate bioprocess optimisation.

Impact of the pentose phosphate pathway on metabolism and pathogenesis of Staphylococcus aureus.

Staphylococcus aureus is an important pathogen that leads to significant disease through multiple routes of infection. We recently published a transposon sequencing (Tn-seq) screen in a mouse acute pneumonia model and identified a hypothetical gene (SAUSA300_1902, pgl) with similarity to a lactonase of Escherichia coli involved in the pentose phosphate pathway (PPP) that was conditionally essential. Limited studies have investigated the role of the PPP in physiology and pathogenesis of S. aureus. We show here that mutation of pgl significantly impacts ATP levels and respiration. RNA-seq analysis of the pgl mutant and parent strains identified compensatory changes in gene expression for glucose and gluconate as well as reductions in the pyrimidine biosynthesis locus. These differences were also evident through unbiased metabolomics studies and 13C labeling experiments that showed mutation of pgl led to reductions in pyrimidine metabolism including decreases in ribose-5P, UMP and GMP. These nucleotide reductions impacted the amount of extracellular DNA in biofilms and reduced biofilm formation. Mutation also limited the capacity of the strain to resist oxidant damage induced by hydrogen peroxide and paraquat and subsequent intracellular survival inside macrophages. Changes in wall teichoic acid impacted susceptibility to hydrogen peroxide. We demonstrated the importance of these changes on virulence in three different models of infection, covering respiratory, skin and septicemia, demonstrating the need for proper PPP function in all models. This work demonstrates the multifaceted role metabolism can play in multiple aspects of S. aureus pathogenesis.

Metabolic reprogramming and altered cell envelope characteristics in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells.

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

ATP13A2 activates the pentose phosphate pathway to promote colorectal cancer growth though TFEB-PGD axis.

BACKGROUND: The pentose phosphate pathway (PPP) is an important mechanism by which tumour cells resist stressful environments and maintain malignant proliferation. However, the mechanism by which the PPP regulates these processes in colorectal cancer (CRC) remains elusive. METHODS: Closely related PPP genes were obtained from the TCGA and GEO databases. The effect of ATP13A2 on CRC cell proliferation was evaluated by performing in vitro assays. The connection between the PPP and ATP13A2 was explored by assessing proliferation and antioxidative stress. The molecular mechanism by which ATP13A2 regulates the PPP was investigated using chromatin immunoprecipitation and dual luciferase experiments. The clinical therapeutic potential of ATP13A2 was explored using patient-derived xenograft (PDX), patient-derived organoid (PDO) and AOM/DSS models. FINDINGS: We identified ATP13A2 as a novel PPP-related gene. ATP13A2 deficiency inhibited CRC growth and PPP activity, as manifested by a decrease in the levels of PPP products and an increase in reactive oxygen species levels, whereas ATP13A2 overexpression induced the opposite effect. Mechanistically, ATP13A2 regulated the PPP mainly by affecting phosphogluconate dehydrogenase (PGD) mRNA expression. Subsequent studies showed that ATP13A2 overexpression promoted TFEB nuclear localization by inhibiting the phosphorylation of TFEB, thereby enhancing the transcription of PGD and ultimately affecting the activity of the PPP. Finally, ATP13A2 knockdown inhibited CRC growth in PDO and PDX models. ATP13A2- /- mice had a lower CRC growth capacity than ATP13A2+/+ in the AOM/DSS model.Our findings revealed that ATP13A2 overexpression-driven dephosphorylation of TFEB promotes PPP activation by increasing PGD transcription, suggesting that ATP13A2 may serve as a potential target for CRC therapy.

LINC01615 maintains cell survival in adaptation to nutrient starvation through the pentose phosphate pathway and modulates chemosensitivity in colorectal cancer.

Numerous mechanisms involved in promoting cancer cell survival under nutrient starvation have been described. Long noncoding RNAs (lncRNAs) have emerged as critical players in colorectal cancer (CRC) progression, but the role of lncRNAs in the progression of CRC under nutrient starvation has not been well clarified. Here, we identified a lncRNA, LINC01615, that was significantly upregulated in response to serum starvation. LINC01615 can contribute to the adaptation of CRC cells to serum-deprived conditions and enhance cell survival under similar conditions. LINC01615 activated the pentose phosphate pathway (PPP) under serum starvation, manifested as decreased ROS production and enhanced nucleotide and lipid synthesis. Glucose-6-phosphate dehydrogenase (G6PD) is a key rate-limiting enzyme of the PPP, and LINC01615 promoted G6PD expression by competitively binding with hnRNPA1 and facilitating G6PD pre-mRNA splicing. Moreover, we also found that serum starvation led to METTL3 degradation by inducing autophagy, which further increased the stability and level of LINC01615 in a m6A-dependent manner. LINC01615 knockdown combined with oxaliplatin achieved remarkable antitumor effects in PDO and PDX models. Collectively, our results demonstrated a novel adaptive survival mechanism permitting tumor cells to survive under limiting nutrient supplies and provided a potential therapeutic target for CRC.

Elevated pentose phosphate pathway flux supports appendage regeneration.

A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.

Ketogenic diet attenuates post-cardiac arrest brain injury by upregulation of pentose phosphate pathway-mediated antioxidant defense in a mouse model of cardiac arrest.

OBJECTIVE: The aim of this study was to investigate the effect of the ketogenic diet (KD) on post-cardiac arrest brain injury in a mouse model of cardiac arrest and cardiopulmonary resuscitation. METHODS: Mice were fed a KD for 4 wk and then subjected to cardiac arrest and cardiopulmonary resuscitation. The HT22 cells after ß-hydroxybutyrate (ß-OHB) treatment were exposed to oxygen-glucose deprivation/reoxygenation. Survival and neurologic function were measured after return of spontaneous circulation. Positron emission tomography/computed tomography scanning, 13C-magnetic resonance spectroscopy analysis, and seahorse assay were performed to explore the mechanism underlying the phenotype. RESULTS: Results of this study demonstrated that KD improved neurologic function and reduced apoptotic neurons in cardiac arrest and cardiopulmonary resuscitation mice. With no alteration of glucose uptake, KD suppressed glucose oxidation in mouse brain. Consistently, the glycolytic capacity of the HT22 cells was also downregulated by ß-OHB treatment. Moreover, KD increased nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate and reduced glutathione/oxidized glutathione couples and reduced reactive oxygen species in the brain, probably due to activation of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway. Pharmacologic inhibition of pentose phosphate pathway totally abolished the influence of ß-OHB on glycolysis, and post-oxygen-glucose deprivation/reoxygenation cell viability and reactive oxygen species production in HT22 cells. CONCLUSION: KD improved survival and attenuated post-cardiac arrest brain injury, which was mediated by upregulation of pentose phosphate pathway and related antioxidant defense system.

N6-methyladenosine modification of circ_0003215 suppresses the pentose phosphate pathway and malignancy of colorectal cancer through the miR-663b/DLG4/G6PD axis.

Circular RNAs (circRNAs) are a recently discovered kind of regulatory RNAs that have emerged as critical biomarkers of various types of cancers. Metabolic reprogramming has gradually been identified as a distinct hallmark of cancer cells. The pentose phosphate pathway (PPP) plays an indispensable role in satisfying the bioenergetic and biosynthetic demands of cancer cells. However, little is known about the role of circRNAs and PPP in colorectal cancer (CRC). The novel circ_0003215 was identified at low levels in CRC and was negatively correlated with larger tumor size, higher TNM stage, and lymph node metastasis. The decreased level of circ_0003215 was resulted from the RNA degradation by m6A writer protein YTHDF2. A series of functional assays demonstrated that circ_0003215 inhibited cell proliferation, migration, invasion, and CRC tumor metastasis in vivo and in vitro. Moreover, circ_0003215 regulated the expression of DLG4 via sponging miR-663b, thereby inducing the metabolic reprogramming in CRC. Mechanismly, DLG4 inhibited the PPP through the K48-linked ubiquitination of glucose-6-phosphate dehydrogenase (G6PD). Taken together, we have identified m6A-modified circ_0003215 as a novel regulator of metabolic glucose reprogramming that inhibited the PPP and the malignant phenotype of CRC via the miR-663b/DLG4/G6PD axis.

Non-oxidative pentose phosphate pathway controls regulatory T cell function by integrating metabolism and epigenetics.

Regulatory T (Treg) cells are critical for maintaining immune homeostasis and preventing autoimmunity. Here, we show that the non-oxidative pentose phosphate pathway (PPP) regulates Treg function to prevent autoimmunity. Deletion of transketolase (TKT), an indispensable enzyme of non-oxidative PPP, in Treg cells causes a fatal autoimmune disease in mice, with impaired Treg suppressive capability despite regular Treg numbers and normal Foxp3 expression levels. Mechanistically, reduced glycolysis and enhanced oxidative stress induced by TKT deficiency triggers excessive fatty acid and amino acid catabolism, resulting in uncontrolled oxidative phosphorylation and impaired mitochondrial fitness. Reduced α-KG levels as a result of reductive TCA cycle activity leads to DNA hypermethylation, thereby limiting functional gene expression and suppressive activity of TKT-deficient Treg cells. We also find that TKT levels are frequently downregulated in Treg cells of people with autoimmune disorders. Our study identifies the non-oxidative PPP as an integrator of metabolic and epigenetic processes that control Treg function.

Enhanced pentose phosphate pathway activity promotes pancreatic ductal adenocarcinoma progression via activating YAP/MMP1 axis under chronic acidosis.

Background: Acidic microenvironment is a common physiological phenomenon in tumors, and is closely related to cancer development, but the effects of acidosis on pancreatic adenocarcinoma (PDAC) remains to be elucidated. Methods: Metabonomic assay and transcriptomic microarray were used to detect the changes of metabolites and gene expression profile respectively in acidosis-adapted PDAC cells. Wound healing, transwell and in vivo assay were applied to evaluate cell migration and invasion capacity. CCK8 and colony formation assays were performed to determine cell proliferation. Results: The acidosis-adapted PDAC cells had stronger metastasis and proliferation ability compared with the control cells. Metabonomic analysis showed that acidosis-adapted PDAC cells had both increased glucose and decreased glycolysis, implying a shift to pentose phosphate pathway. The metabolic shift further led to the inactivation of AMPK by elevating ATP. Transcriptomic analysis revealed that the differentially expressed genes in acidosis-adapted cells were enriched in extracellular matrix modification and Hippo signaling. Besides, MMP1 was the most upregulated gene in acidosis-adapted cells, mediated by the YAP/TAZ pathway, but could be reduced by AMPK activator. Conclusion: The present study showed that metabolic reprogramming promotes proliferation and metastasis of acidosis-adapted PDAC cells by inhibiting AMPK/Hippo signaling, thus upregulating MMP1.

TKTL1 Knockdown Impairs Hypoxia-Induced Glucose-6-phosphate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase Overexpression.

Increased expression of transketolase (TKT) and its isoform transketolase-like-1 (TKTL1) has been related to the malignant leukemia phenotype through promoting an increase in the non-oxidative branch of the pentose phosphate pathway (PPP). Recently, it has also been described that TKTL1 can have a role in survival under hypoxic conditions and in the acquisition of radio resistance. However, TKTL1's role in triggering metabolic reprogramming under hypoxia in leukemia cells has never been characterized. Using THP-1 AML cells, and by combining metabolomics and transcriptomics techniques, we characterized the impact of TKTL1 knockdown on the metabolic reprogramming triggered by hypoxia. Results demonstrated that TKTL1 knockdown results in a decrease in TKT, glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities and impairs the hypoxia-induced overexpression of G6PD and GAPDH, all having significant impacts on the redox capacity of NADPH- and NADH-related cells. Moreover, TKTL1 knockdown impedes hypoxia-induced transcription of genes encoding key enzymes and transporters involved in glucose, PPP and amino acid metabolism, rendering cells unable to switch to enhanced glycolysis under hypoxia. Altogether, our results show that TKTL1 plays a key role in the metabolic adaptation to hypoxia in THP-1 AML cells through modulation of G6PD and GAPDH activities, both regulating glucose/glutamine consumption and the transcriptomic overexpression of key players of PPP, glucose and amino acids metabolism.

NADPH production in dark stages is critical for cyanobacterial photocurrent generation: a study using mutants deficient in oxidative pentose phosphate pathway.

Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected to be correlated with the redox environment of the photosynthetic cells, the relationship between the photocurrent and the cellular redox state is poorly understood. Here, we investigated the effect of the reduced nicotinamide adenine dinucleotide phosphate [NADP(H)] redox level of cyanobacterial cells (before light exposure) on the photocurrent using several mutants (Δzwf, Δgnd, and ΔglgP) deficient in the oxidative pentose phosphate (OPP) pathway, which is the metabolic pathway that produces NADPH in darkness. The NAD(P)H redox level and photocurrent in the cyanobacterium Synechocystis sp. PCC 6803 were measured noninvasively. Dysfunction of the OPP pathway led to oxidation of the photosynthetic NADPH pool in darkness. In addition, photocurrent induction was retarded and the current density was lower in Δzwf, Δgnd, and ΔglgP than in wild-type cells. Exogenously added glucose compensated the phenotype of ΔglgP and drove the OPP pathway in the mutant, resulting in an increase in the photocurrent. The results indicated that NADPH accumulated by the OPP pathway before illumination is a key factor for the generation of a photocurrent. In addition, measuring the photocurrent can be a non-invasive approach to estimate the cellular redox level related to NADP(H) pool in cyanobacteria.

Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken-egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a "metabolism first" theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

Enhancement of riboflavin production in Bacillus subtilis via in vitro and in vivo metabolic engineering of pentose phosphate pathway.

OBJECTIVES: The production of riboflavin with Bacillus subtilis, is an established process, however it is yet to be fully optimized. The aim of this study was to explore how riboflavin yields can be improved via in vitro and in vivo metabolic engineering modification of the pentose phosphate pathway (PPP). RESULTS: In vitro, glucose was replaced with sodium gluconate to enhance PPP. Flask tests showed that the riboflavin titer increased from 0.64 to 0.87 g/L. The results revealed that the direct use of sodium gluconate could benefit riboflavin production. In vivo, gntP (encoding gluconate permease) was overexpressed to improve sodium gluconate uptake. The riboflavin titer reached 1.00 g/L with the mutant B. subtilis RF01. Ultimately, the fermentation verification of the engineered strain was carried out in a 7-L fermenter, with the increased riboflavin titer validating this approach. CONCLUSIONS: The combination of metabolic engineering modifications in vitro and in vivo was confirmed to promote riboflavin production efficiently by increasing PPP and has great potential for industrial application. This work is aimed to explore how to improve the riboflavin yield by the rational renovation of the pentose phosphate pathway (PPP). In vitro, metabolic engineering mainly uses sodium gluconate as a carbon source instead of glucose, and in vivo, metabolic engineering mainly includes the overexpression of sodium gluconate utility-related genes. The effect of sodium gluconate on cell growth, riboflavin production was investigated in the flasks and fermenter scale.

Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis.

Background: Clear cell renal cell carcinoma (ccRCC) is characterized by glycogen-laden, unexplained male predominance, and frequent mutations in the Von Hippel-Lindau (VHL) gene and histone modifier genes. Besides, poor survival rates of ccRCC patients seem to be associated with up-regulation of the pentose phosphate pathway (PPP). However, the mechanism underlying these features remains unclear. Methods: Whole exome sequencing was used to identify the gene mutation that implicated in the rewired glucose metabolism. RNA-seq analyses were performed to evaluate the function of KDM5C in ccRCC. Furthermore, heavy isotope tracer analysis and metabolites quantification assays were used to study how KDM5C affects intracellular metabolic flux. To provide more in vivo evidence, we generated the Kdm5c-/- mice by CRISPR-Cas9 mediated gene knockout and performed the xenografts with KDM5C overexpressing or depleted cell lines. Results: A histone demethylase gene KDM5C, which can escape from X-inactivation and is predominantly mutated in male ccRCC patients, was identified to harbor the frameshift mutation in the ccRCC cell line with the highest glycogen level, while the restoration of KDM5C significantly reduced the glycogen level. Transcriptome and metabolomic analysis linked KDM5C to metabolism-related biological processes. KDM5C specifically regulated the expression of several hypoxia-inducible factor (HIF)-related genes and Glucose-6-phosphate dehydrogenase (G6PD) that were involved in glycogenesis/glycogenolysis and PPP, respectively, mainly through the histone demethylase activity of KDM5C. Depletion of KDM5C increased the production of glycogen, which was then directed to glycogenolysis to generate glucose-6-phosphate (G6P) and subsequently PPP to produce nicotinamide adenine dinucleotide phosphate hydride (NADPH) and glutathione (GSH), thus conferring cells resistance to reactive oxygen species (ROS) and ferroptosis. KDM5C re-expression suppressed the glucose flux through PPP and re-sensitized cancer cells to ferroptosis. Notably, Kdm5c-knockout mice kidney tissues exhibited elevated glycogen level, reduced lipid peroxidation and displayed a transformation of renal cysts into hyperplastic lesions, implying a cancer-protective benefit of ferroptosis. Furthermore, KDM5C deficiency predicted the poor prognosis, and clinically relevant KDM5C mutants failed to suppress glycogen accumulation and promoted ferroptosis as wild type. Conclusion: This work revealed that a histone modifier gene inactive mutation reprogramed glycogen metabolism and helped to explain the long-standing puzzle of male predominance in human cancer. In addition, our findings may suggest the therapeutic value of targeting glycogen metabolism in ccRCC.