Ectoine is a highly valuable amino acid derivative with multiple functionalities, which finds extensive applications in cosmetics, pharmaceuticals, and life sciences. Developing microbial strains capable of high-level ectoine production has emerged as a prominent research focus in recent years. Here, we employed a systematic metabolic engineering strategy to transform wild-type Escherichia coli into a high-yield ectoine-producing strain. First, we constructed a heterologous ectoine synthesis pathway in E. coli W3110. By knocking out the bifunctional enzymes ThrA and MetL, as well as LysA to block byproduct formation and overexpressing an optimized LysC as a substitute, we enhanced the supply of the direct precursor. We also investigated the impact of the copy number on ectoine synthesis. Subsequently, we modified the 5' untranslated region of citrate dehydrogenase gltA to fine-tune its expression, balancing cellular growth with product synthesis. To augment glutamate amino donor availability, we heterologously overexpressed Bacillus subtilis gltAB to enhance glutamate supply, while boosting pntAB expression to maintain cofactor equilibrium. Similarly, we fortified the glucose-to-oxaloacetate synthetic pathway through a series of metabolic modifications, achieving a yield of 5.94 g/L in shake flask fermentation. Finally, under controlled batch glucose feeding, strain E20 produced 88.1 g/L of ectoine over a 60 h fermentation period, and a glucose conversion rate of 0.26 g/g. This study employed metabolic engineering strategies to enhance the accumulation of oxaloacetate and utilized 5'-UTR engineering to finely regulate GltA expression, thereby balancing cell growth, These strategies, including increasing aspartate accumulation, enhancing the catalytic efficiency of the key heterologous enzyme LysCpa in the aspartate to aspartate phosphate pathway, and boosting glutamate as an amino donor to enhance the synthesis of aspartate from oxaloacetic acid, can be applied to the synthesis of other amino acids in the aspartate family.

Glucose deprivation is a major metabolic stress that requires coordinated adaptive responses to maintain cellular homeostasis and survival, yet the role of tripartite motif-containing 24 (TRIM24) in this process remains unclear. To address this question, we generated CRISPR-Cas9-mediated TRIM24-knockout MCF-7 and HEK293 cell lines, performed targeted metabolomic profiling and aspartate assays, used 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), aminooxyacetic acid (AOA), aspartate supplementation, and glutamic-oxaloacetic transaminase 2 (GOT2) knockdown to probe AMPK signaling and aspartate metabolism, and examined starvation responses in constitutive Trim24 knockout mice on a C57BL/6 background. Loss of TRIM24 sensitized cells to glucose deprivation. Re-expression of TRIM24 partially restored cell viability under glucose deprivation in both MCF-7 and HEK293 cells. Under glucose-free conditions, TRIM24 deficiency was associated with impaired AMP-activated protein kinase (AMPK) pathway activation, increased intracellular aspartate accumulation, and altered ATP/AMP levels. Pharmacological reactivation of AMPK by AICAR improved the survival of TRIM24-deficient cells under glucose deprivation. Reducing intracellular aspartate by AOA treatment or GOT2 knockdown restored AMPK pathway activation and improved adaptation to glucose deprivation, whereas exogenous aspartate suppressed AMPK signaling and increased ATP/AMP levels. In vivo, starvation of Trim24-deficient mice was associated with reduced AMPK pathway activation and increased aspartate levels. Together, these findings support a model in which TRIM24 contributes to adaptation to glucose deprivation and in which abnormal aspartate accumulation contributes to impaired AMPK pathway activation in TRIM24-deficient cells.

The effects of polyvinyl chloride microplastics and zinc oxide nanoparticles co-exposure on nutritional quality of purple waxy maize grains.

Hepatocellular carcinoma (HCC) progression and therapy sensitivity are critically fueled by liver cancer stem cells (LCSCs), yet the regulatory mechanisms of circular RNAs (circRNAs) on LCSCs remain elusive. Here, through circRNA microarray analysis of LCSCs and non‐stem HCC cells, circRAPGEF1 is identified as a LCSC‐enriched circRNA upregulated in HCC tissues and predictive of poor patient survival. Functionally, circRAPGEF1 promoted the stemness properties, proliferation, and tumorigenicity of HCC cells. Mechanistically, the METTL3‐mediated N6‐methyladenosine (m6A) modification of circRAPGEF1 facilitated KH domain‐dependent binding of IGF2BP3 to its UGGAC motif, which conferring stability to circRAPGEF1 while competitively disrupting the IGF2BP3/ASS1 mRNA interaction. This process led to the degradation of ASS1 mRNA, triggering aspartate accumulation and activation of the S6K/CAD signaling pathway. Crucially, circRAPGEF1 overexpression reduced the sorafenib sensitivity, whereas targeting circRAPGEF1 using nanoparticles‐mediated systematic siRNAs delivery effectively sensitized HCC cells to sorafenib. Collectively, these findings unveil a METTL3/circRAPGEF1/IGF2BP3/ASS1 regulatory axis that drives aspartate metabolic reprogramming to fuel HCC stemness properties, positioning circRAPGEF1 as a dual prognostic biomarker and therapeutic target to enhance sorafenib efficacy in HCC.

Abstract These studies investigate the antitumor effects of targeting serine hydroxymethyltransferase (SHMT) 2 under hypoxia, the downstream metabolic consequences, and how to exploit unique metabolic vulnerabilities for cancer therapy. Mitochondrial one-carbon (C1) flux through SHMT2 and 5, 10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) is the major source of C1 units in cancer cells. In mitochondria, THF serves as a cofactor for the catabolism of serine by SHMT2 to produce glycine and 5, 10-methylene-THF which is subsequently metabolized by MTHFD2 and MTHFD1L. The major products of this mitochondrial C1 flux include formate and NADH, along with glycine for protein, purine and glutathione (GSH) synthesis. Thus, mitochondrial C1 metabolism is important for maintaining redox homeostasis and scavenging reactive oxygen species (ROS). Under hypoxic conditions, the flux through SHMT2 and MTHFD2 is the major source of NADH; therefore, inhibition of SHMT2 under hypoxia may increase oxidative stress resulting in cell death. However, inhibition of SHMT2 under hypoxia may facilitate cell growth as a result of aspartate accumulation due to depletion of NADH and suppression of the TCA cycle. Aspartate accumulation preserves de novo purine biosynthesis under these conditions via donation of an amine group to IMP. We discovered novel 5-substituted pyrrolo[3, 2-d]pyrimidine multitargeted antifolates (e.g., AGF347) which inhibit SHMT2 in the mitochondria in addition to cytosolic targets (SHMT1 and folate-dependent enzymes in purine biosynthesis). We hypothesized that these agents may overcome inherent challenges of targeting SHMT2 under hypoxia, reflecting their inhibition of purine biosynthetic targets to preserve antitumor activity. Further, since all current SHMT2 inhibitors also inhibit SHMT1, to uncover the respective impacts of inhibiting these homologous enzymes, we utilized tetracycline-inducible SHMT1 or SHMT2 HCT116 tumor cell models to simulate selective inhibition of these targets by removal of doxycycline. We demonstrate that the pyrrolo[3, 2-d]pyrimidine compounds retain potent antitumor efficacy under hypoxia, as reflected in inhibition of cell proliferation and colony formation. We assessed the metabolic consequences of antifolate treatment under hypoxia and normoxia by measuring malate, aspartate, and purine levels by LC-MS/MS in addition to effects on ROS and GSH levels. We also demonstrated that loss of SHMT2 activity alone is sufficient for anticancer effects under both normoxia and hypoxia, whereas loss of SHMT1 alone had no effect. These studies demonstrate the considerable promise of multitargeted pyrrolo[3, 2-d]pyrimidine antifolates for cancer therapy under conditions under which standard-of-care agents may be less effective. Citation Format: Mathew Joseph Schneider, Carrie OConnor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Multitargeting of SHMT2 and purine biosynthesis is an effective antitumor treatment under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3101.

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.

Staphylococcus aureus adapts to the immunometabolite itaconic acid by inducing acid and oxidative stress responses including S-bacillithiolations and S-itaconations

Photoreceptor cell death is the cause of vision loss in many forms of retinal disease. Metabolic dysfunction within the outer retina has been shown to be an underlying factor contributing to photoreceptor loss. Therefore, a comprehensive understanding of the metabolic pathways essential to photoreceptor health and function is key to identifying novel neuroprotective strategies. Glutamic-oxaloacetic transaminase 1 (Got1) encodes for a cytosolic aspartate aminotransferase that reversibly catalyzes the transfer of an amino group between glutamate and aspartate and is an important aspect of the malate-aspartate shuttle (MAS), which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Previous work has demonstrated that the activity of this enzyme is highest in photoreceptor inner segments. Furthermore, ex vivo studies have demonstrated that the retina relies on aspartate aminotransferase for amino acid metabolism. Importantly, aspartate aminotransferase has been suggested to be an early biomarker of retinal degeneration in retinitis pigmentosa and a possible target for neuroprotection. In the present study, we characterized the effect of Got1 deletion on photoreceptor metabolism, function, and survival in vivo by using a rod photoreceptor-specific, Got1 knockout mouse model. Loss of the GOT1 enzyme from rod photoreceptors resulted in age-related photoreceptor degeneration with an accumulation of retinal aspartate and NADH and alterations in the expression of genes involved in the MAS, the tricarboxylic acid (TCA) cycle, and redox balance. Hence, GOT1 is critical to in vivo photoreceptor metabolism, function, and survival.

SummaryCastration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge (Oakes and Papa, 2015). Matched untargeted multi-level omic datasets may reveal biological changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics were performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node, and bone metastasis grown as murine orthografts. Collectively, we tested the feasibility of muti-omics analysis on models of CRPC in revealing pathways of interest for future validation investigation. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-acetylated alpha-linked acidic dipeptidases (FOLH1/NAALADL2). Based on pathway analysis integrating multiple omic levels, we hypothesize that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of acetyl Co-A for upregulated sphingolipid metabolism and a pool of glutamate and aspartate for nucleotide synthesis during tumor growth.

3207 – UNDERSTANDING AND TARGETING ASPARTATE METABOLISM IN THE LEUKEMIC NICHE

Inhibition of glucose transport synergizes with chemical or genetic disruption of mitochondrial metabolism and suppresses TCA cycle-deficient tumors

The inoculation response of single arbuscular mycorrhiza fungi (AMF) or rhizobia (Rh) in relation to nitrogen (N) acquisition of plants is well established, while the combined effect of both AMF and Rh is poorly known with regard to N assimilation for changes in amino acids of white clover (Trifolium repens). A pot study was carried out to evaluate the effect of single versus dual inoculation of AMF (Rhizophagus intraradices) and rhizobium (Rhizobium trifolii) on plant growth, leaf and root N contents, root amino acid contents, and root N-related enzyme activities in white clover. One hundred days after inoculations, Rh inoculation significantly stimulated the root colonization by R. intraradices. A single inoculation of AMF or Rh improved the plant growth (biomass production, root projected area, and root volume), root N acquisition, and dual inoculation of AMF and Rh further expanded some of these positive effects on root projected area and root N contents than single inoculation. All the inoculations notably increased activities of root asparagines synthase, nitrate reductase, and glutamate synthase, whilst dual inoculation displayed a much stronger effect in asparagine synthase activity than single inoculation. Single Rh treatment increased root glutamate and proline content, single AMF inoculation induced an increase in glutamate, aspartate, arginine, and ornithine content, while dual inoculation stimulated the accumulation of aspartate and proline. These results suggested the cooperation between the AMF and Rh inoculations, which magnified the positive effect on partly N metabolites and N-assimilation relevant enzymes of white clover.

Multi-Omics Analysis Identifies Local N-Acetyl Aspartate Accumulation as a Feature of Castration Resistant Prostate Cancer

Asthenozoospermia (AS), defined as low-motility spermatozoa in the ejaculate, is a frequent cause of human male infertility. DJ-1 (also known as PARK7), a protein highly associated with male sterility, binds to the mitochondrial complex I subunit to protect mitochondrial function. However, its involvement in spermatogenesis has not been fully elucidated. Previously, the levels of DJ-1 were shown to be significantly decreased in testicular tissues of rats with ornidazole (ORN)-induced AS. Here, we used a rat model to investigate the localization and expression levels of DJ-1 and its interacting NDUFS3 and NDUFA4 mitochondrial complex I subunits, as well as AS-induced metabolic alterations in testicular tissues. ORN significantly reduced the levels of DJ-1 in the nucleus of secondary spermatocytes, while increasing the expression of NDUFS3 in the cytoplasm of primary spermatocytes. Further, NDUFA4 showed higher expression after treatment with ORN. The principal ORN-induced changes in metabolic small molecules related to the accumulation of glucose, glutamine, and N-acetyl aspartate, enhancement of purine pathway, increase of the phosphatidic acid (PA) (18:0/18:1), phosphatidylethanolamine (PE) (16:0/18:1), and PA (18:0/20:4) lipid metabolites, and imbalance in the concentrations of Na+ and K+. However, we did not observe any abnormalities of certain small metabolic molecules and metal ions in semen samples from patients with AS. In conclusion, these results suggest that DJ-1 deficiency in testicular tissues might be closely related to the localization of NDUFS3 and content of NDUFA4, thus causing abnormalities in the mitochondrial energy metabolism and multiple other metabolic pathways.

Physiological and metabolic analysis showed the difference in drought tolerance and revealed the common and specific metabolic regulations in Salix sinopurpurea and Salix suchowensis. Willows (Salix spp.) are important woody plants as promising sources for sustainable and renewable biomass. However, drought is highly detrimental to their growth and development. Deciphering the adaptation mechanism of willows to drought will provide a theoretical basis for the cultivation of drought-tolerant varieties. In this work, we investigated the physiological and metabolic responses to drought stress in Salix sinopurpurea and Salix suchowensis. The drought experiment was conducted on clonal plants from cuttings of the two willow species under greenhouse conditions. S. sinopurpurea exhibited higher drought tolerance, as evidenced by lower growth reduction, and higher leaf relative water content, water use efficiency and net photosynthesis rate than those of S. suchowensis under the same drought conditions. Metabolic profiling identified 67 and 64 differentially accumulated metabolites in S. sinopurpurea and S. suchowensis, respectively. These metabolites function as compatible solutes and energy reserves and in antioxidant protection to defend against drought stress. Carbohydrate, amino acid and lipid metabolic pathways were central in the metabolic regulations of the drought response in the two willow species. The accumulation of aspartate, glutamate, serine, threonine, and sedoheptulose particularly in S. sinopurpurea might equip this species with drought tolerance. Meanwhile, phenylalanine and phytosterols were specifically inhibited in S. suchowensis, which might be associated with its susceptibility to drought stress. Taken together, these results provide a framework for better understanding the metabolic responses of willow to drought stress.

Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Changes in the levels of plant metabolites in response to nutrient deficiency is indicative of how plants utilize scarce resources. In this study, changes in the metabolite profile of roots and shoots of wheat genotypes differing in phosphorus use efficiency (PUE) was investigated. Under low P supply and at 28 days after sowing (DAS), the wheat breeding line, RAC875 (P efficient) produced 42% more shoot biomass than the wheat variety, and Wyalkatchem (P inefficient). Significant changes in the metabolite profile in leaves and roots were observed under low P supply and significant genotypic variation was evident. Under low P supply, an increase in raffinose and 1-kestose was evident in roots of both wheat genotypes, with RAC875 accumulating more when compared to Wyalkatchem. There was no significant increase in raffinose and 1-kestose in leaves when plants were grown under P deficiency. P deficiency had no significant impact on the levels of sucrose, maltose, glucose and fructose in both genotypes, and while phosphorylated sugars (glucose-6-P and fructose-6-P) remained unchanged in RAC875, in Wyalkatchem, glucose-6-P significantly decreased in roots, and fructose-6-P significantly decreased in both leaves and roots. Glycerol-3-P decreased twofold in roots of both wheat genotypes in response to low P. In roots, RAC875 exhibited significantly lower levels of fumarate, malate, maleate and itaconate than Wyalkatchem, while low P enhanced organic acid exudation in RAC875 but not in Wyalkatchem. RAC875 showed greater accumulation of aspartate, glutamine and β-alanine in leaves than Wyalkatchem under low P supply. Greater accumulation of raffinose and 1-kestose in roots and aspartate, glutamine and β-alanine in leaves appears to be associated with enhanced PUE in RAC875. Glucose-6-P and fructose-6-P are important for glycolysis, thus maintaining these metabolites would enable RAC875 to maintain carbohydrate metabolism and shoot biomass under P deficiency. The work presented here provides evidence that differences in metabolite profiles can be observed between wheat varieties that differ in PUE and key metabolic pathways are maintained in the efficient genotype to ensure carbon supply under P deficiency.

Failure in intracellular zinc accumulation is a key process in prostate carcinogenesis. Nevertheless, epidemiological studies of zinc administration have provided contradicting results. In order to examine the impact of the artificial intracellular increase of zinc(II) ions on prostate cancer metabolism, PNT1A, 22Rv1, and PC-3 prostatic cell lines-depicting different stages of cancer progression-and their zinc-resistant counterparts were used. To determine "benign" and "malignant" metabolic profiles, amino acid patterns, gene expression, and antioxidant capacity of these cell lines were assessed. Amino acid profiles were examined using an ion-exchange liquid chromatography. Intracellular zinc content was measured by atomic absorption spectrometry. Metallothionein was quantified using differential pulse voltammetry. The content of reduced glutathione was determined using high performance liquid chromatography coupled with an electrochemical detector. Cellular antioxidant capacity was determined by the ABTS test and gene expression analysis was performed by qRT-PCR. Long-term zinc treatment was shown to reroute cell metabolism from benign to more malignant type. Long-term application of high concentration of zinc(II) significantly enhanced cisplatin resistance, invasiveness, cellular antioxidant capacity, synthesis of glutathione, and expression of treatment resistance- and stemness-associated genes (SOX2, POU5F1, BIRC5). Tumorous cell lines universally displayed high accumulation of aspartate and sarcosine and depletion of essential amino acids. Increased aspartate/threonine, aspartate/methionine, and sarcosine/serine ratios were associated with cancer phenotype with high levels of sensitivity and specificity. Prostate 77: 604-616, 2017. © 2017 Wiley Periodicals, Inc.

Metabolomic responses of Haliotis diversicolor to organotin compounds

We studied the effect of thrombosis of retina vessels on the development of glutamate excitotoxicity in chinchilla rabbits. Thrombosis was induced by administration of Bengal rose dye at a dose of 40 mg/kg into the auricular vein of the animals followed by focal lighting with white light. Lighting was performed for 10 min on average at the temporal vascular arcade in the location of the angulation of vessels over the border of the disk of the optic nerve using a binocular ophtalmoscope and a condensing +14.0 D lens. We found that experimental thrombosis induced the accumulation of glutamate, aspartate, glycine, and GABA, as well as oxidative stress, which was associated with decreased activity of superoxide dismutase, glutathione peroxidase, and glutathione-S-transferase.