Purine Biosynthesis Pathway Research Articles

The metabolic engineering of Escherichia coli for the high-yield production of hypoxanthine

BackgroundHypoxanthine, prevalent in animals and plants, is used in the production of food additives, nucleoside antiviral drugs, and disease diagnosis. Current biological fermentation methods synthesize quantities insufficient to meet industrial demands. Therefore, this study aimed to develop a strain capable of industrial-scale production of hypoxanthine.ResultsDe novo synthesis of hypoxanthine was achieved by blocking the hypoxanthine decomposition pathway, thus alleviating transcriptional repression and multiple feedback inhibition, and introducing a purine operon from Bacillus subtilis to construct a chassis strain. The effects of knocking out the IMP(Inosine 5’-monophosphate) branch on the growth status and titer of the strain were then investigated, and the effectiveness of adenosine deaminase and adenine deaminase was verified. Overexpressing these enzymes created a dual pathway for hypoxanthine synthesis, enhancing the metabolic flow of hypoxanthine synthesis and preventing auxotrophic strain formation. Introducing IMP-specific 5’ -nucleotidase addressed the issue of adenylate accumulation. In addition, the metabolic flow of the guanine branch was dynamically regulated by the guaB gene. The supply of glutamine and aspartic acid precursors was enhanced by introducing an exogenous glnA mutant gene, overexpressing aspC, and replacing the weaker promoter to regulate the aspartic acid branching pathway. Ultimately, fermentation in a 5 L bioreactor for 48 h produced 30.6 g/L hypoxanthine, with a maximum real-time productivity of 1.4 g/L/h, the highest value of hypoxanthine production by microbial fermentation reported so far.ConclusionsThe intracellular purine biosynthesis pathway is extensive and regulated at multiple levels in cells. The IMP branch in the hypoxanthine synthesis pathway has a higher metabolic flux. The current challenge lies in systematically allocating the metabolic flux within the branch pathway to achieve substantial product accumulation. In this study, E. coli was used as the chassis strain to construct a dual pathway for IMP and AMP(Adenosine 5’-monophosphate) synergistic hypoxanthine synthesis and dynamically regulate the guanine branch pathway. Overall, our experimental efforts culminated in a high-yield, plasmid- and defect-free engineered hypoxanthine strain.

Systemic inhibition of de novo purine biosynthesis prevents weight gain and improves metabolic health by increasing thermogenesis and decreasing food intake.

Obesity is a major health concern, largely because it contributes to type 2 diabetes mellitus (T2DM), cardiovascular disease, and various malignancies. Increase in circulating amino acids and lipids, in part due to adipose dysfunction, have been shown to drive obesity-mediated diseases. Similarly, elevated purines and uric acid, a degradation product of purine metabolism, are found in the bloodstream and in adipose tissue. These metabolic changes are correlated with metabolic syndrome, but little is known about the physiological effects of targeting purine biosynthesis. To determine the effects of purine biosynthesis on organismal health we treated mice with mizoribine, an inhibitor of inosine monophosphate dehydrogenase 1 and 2 (IMPDH1/2), key enzymes in this pathway. Mice were fed either a low-fat (LFD; 13.5% kcal from fat) or a high-fat (HFD; 60% kcal from fat) diet for 30 days during drug or vehicle treatment. We ascertained the effects of mizoribine on weight gain, body composition, food intake and absorption, energy expenditure, and overall metabolic health. Mizoribine treatment prevented mice on a HFD from gaining weight, but had no effect on mice on a LFD. Body composition analysis demonstrated that mizoribine significantly reduced fat mass but did not affect lean mass. Although mizoribine had no effect on lipid absorption, food intake was reduced. Furthermore, mizoribine treatment induced adaptive thermogenesis in skeletal muscle by upregulating sarcolipin, a regulator of muscle thermogenesis. While mizoribine-treated mice exhibited less adipose tissue than controls, we did not observe lipotoxicity. Rather, mizoribine-treated mice displayed improved glucose tolerance and reduced ectopic lipid accumulation. Inhibiting purine biosynthesis prevents mice on a HFD from gaining weight, and improves their metabolic health, to a significant degree. We also demonstrated that the purine biosynthesis pathway plays a previously unknown role in skeletal muscle thermogenesis. A deeper mechanistic understanding of how purine biosynthesis promotes thermogenesis and decreases food intake may pave the way to new anti-obesity therapies. Crucially, given that many purine inhibitors have been FDA-approved for use in treating various conditions, our results indicate that they may benefit overweight or obese patients. A purine biosynthesis inhibitor, mizoribine, protects against diet-induced weight gainMizoribine prevents fat mass gain in high-fat diet-fed male miceMizoribine reduces food intake and increases thermogenesisMizoribine induces expression of sarcolipin, a regulator of thermogenesisMizoribine treatment reduces ectopic lipids and increases glucose tolerance.

Effects of Lactocaseibacillus paracasei subsp. paracasei NTU 101 on gut microbiota: a randomized, double-blind, placebo-controlled clinical study.

Lactocaseibacillus paracasei subsp. paracasei NTU 101 (NTU101) is a well-known commercial probiotic with multiple health beneficial effects. In this study, the gut microbiota modulation effect of an NTU 101 product, Vigiis 101-LAB, on healthy human was investigated in a randomized, double-blind, placebo-controlled human trial. Vigiis 101-LAB significantly modulated human gut microbiota at fourth and sixth weeks of trial (anosim analysis, P = 0.001). It also significantly improved peristalsis (P = 0.003) and shortened defecation interval of subjects. The shift of gut microbiota is significantly fit with defecation interval (P = 0.009) and stool shape (P = 0.001) of subjects. Our results suggest that Vigiis 101-LAB promotes human intestinal health with improvement of peristalsis and fecal quality. The gut modulation effects of Vigiis 101-LAB subsequently raised the abundance of vitamin B7, vitamin K, pyrimidine and purine biosynthesis pathways. Vigiis 101-LAB may promote peristalsis via purinergic pathway and possibly conferring prophylactic benefits against irritable bowel syndrome with constipation. © 2024 Society of Chemical Industry.

De novo engineering riboflavin production Bacillus subtilis by overexpressing the downstream genes in the purine biosynthesis pathway

BackgroundBacillus subtilis is widely used in industrial-scale riboflavin production. Previous studies have shown that targeted mutagenesis of the ribulose 5-phosphate 3-epimerase in B. subtilis can significantly enhance riboflavin production. This modification also leads to an increase in purine intermediate concentrations in the medium. Interestingly, B. subtilis exhibits remarkable efficiency in purine nucleoside synthesis, often exceeding riboflavin yields. These observations highlight the importance of the conversion steps from inosine-5’-monophosphate (IMP) to 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone-5’-phosphate (DARPP) in riboflavin production by B. subtilis. However, research elucidating the specific impact of these reactions on riboflavin production remains limited.ResultWe expressed the genes encoding enzymes involved in these reactions (guaB, guaA, gmk, ndk, ribA) using a synthetic operon. Introduction of the plasmid carrying this synthetic operon led to a 3.09-fold increase in riboflavin production compared to the control strain. Exclusion of gmk from the synthetic operon resulted in a 36% decrease in riboflavin production, which was further reduced when guaB and guaA were not co-expressed. By integrating the synthetic operon into the genome and employing additional engineering strategies, we achieved riboflavin production levels of 2702 mg/L. Medium optimization further increased production to 3477 mg/L, with a yield of 0.0869 g riboflavin per g of sucrose.ConclusionThe conversion steps from IMP to DARPP play a critical role in riboflavin production by B. subtilis. Our overexpression strategies have demonstrated their effectiveness in overcoming these limiting factors and enhancing riboflavin production.

Abstract PO5-23-08: Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells

Abstract Eighty percent of breast cancers express estrogen receptor alpha (ERα, ESR1) at the time of diagnosis. Endocrine therapy (ET) is the standard treatment that either block estrogen mediated ER activation (such as tamoxifen, fulvestrant) or suppress estrogen synthesis (such as exemestane, letrozole, or anastrozole). While ET is initially effective, emergence of ET-resistance is common and possess a major clinical challenge. Hence, there is an ongoing need to develop new treatments for ET-resistant-ERα positive breast cancer. In this study we investigated the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway as a potential target of ET-resistant breast cancer. The protein level of ADSL in two independent ET-resistant cell line model LCC9 and T47D-4HT cells were significantly higher in comparison to its parental ET-sensitive cell lines, MCF7 and T47D cells, respectively. Transient knockdown of ADSL using two independent siRNA in both LCC9 and T47D-4HT cells results in a significant decrease of cell growth, colony, and spheroid formation. Notably, reduced ADSL levels in LCC9 cells prevented the cells from progressing from G1 to S phase of cell cycle with concurrent elevation of cyclin D1/D3, CDK2/4, and cyclin E. On the other hand, in T47D-4HT cells, ADSL reduction caused cells to accumulate in S-phase of cell cycle and showed lower levels of total cyclin D1 protein. Mechanistically, in LCC9 cells (but not in T47D-4HT cells), ADSL depletion induced DNA replication stress which activates (phosphorylate) ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (Chk1) that in turn failed to de-phosphorylate the inhibitory-phospho-groups of cyclin-dependent kinase 2 (CDK2). In addition, ADSL depletion perturbed the mitochondrial function in both LCC9 and T47D-4HT cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was lower in both the cells along with the mitochondrial membrane potential. Pertinently, lower concentration of AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), a product of ADSL enzyme function, partially rescued the cell growth and restored mitochondrial membrane potential in ADSL-depleted LCC9 cells. To further understand the global effects of ADSL depletion we performed RNA-sequencing of LCC9 and T47D-4HT cells after ADSL depletion and compared with respective control siRNA transfected cells followed by gene set enrichment analysis. Intriguingly, diverse effect was observed in LCC9 and T47D-4HT cells as very few genes were found to be commonly regulated between these two cell lines. Analysis of clinical data sets revealed high level of ADSL transcript was associated with adverse progression free survival and overall survival in breast cancer patients treated with endocrine therapies. Overall, our findings demonstrate that ADSL-mediated de novo purine synthesis is critical for cellular growth, proliferation, and mitochondrial function of endocrine therapy-resistant breast cancer cells. Therefore, targeting ADSL is a novel potential therapeutic approach for ERα positive ET- resistant breast cancer. Citation Format: Anil Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-08.

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1985.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

Purine Biosynthesis Pathways Are Required for Myogenesis in Xenopus laevis.

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase (ADSL), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat, which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.

Volatile anesthetic isoflurane exposure facilitates Enterococcus biofilm infection.

Enterococcus faecalis (E. faecalis) is one of the major pathogenic bacteria responsible for surgical site infections. Biofilm infections are major hospital-acquired infections. Previous studies suggested that ions could regulate biofilm formation in microbes. Volatile anesthetics, frequently administered in surgical setting, target ion channels. Here, we investigated the role of ion channels/transporters and volatile anesthetics in the biofilm formation by E. faecalis MMH594 strain and its ion transporter mutants. We found that a chloride transporter mutant significantly reduced biofilm formation compared to the parental strain. Downregulation of teichoic acid biosynthesis in the chloride transporter mutant impaired biofilm matrix formation and cellular adhesion, leading to mitigated biofilm formation. Among anesthetics, isoflurane exposure enhanced biofilm formation in vitro and in vivo. The upregulation of de novo purine biosynthesis pathway by isoflurane exposure potentially enhanced biofilm formation, an essential process for DNA, RNA, and ATP synthesis. We also demonstrated that isoflurane exposure to E. faecalis increased cyclic-di-AMP and extracellular DNA production, consistent with the increased purine biosynthesis. We further showed that isoflurane enhanced the enzymatic activity of phosphoribosyl pyrophosphate synthetase (PRPP-S). With the hypothesis that isoflurane directly bound to PRPP-S, we predicted isoflurane binding site on it using rigid docking. Our study provides a better understanding of the underlying mechanisms of E. faecalis biofilm formation and highlights the potential impact of an ion transporter and volatile anesthetic on this process. These findings may lead to the development of novel strategies for preventing E. faecalis biofilm formation and improving patient outcomes in clinical settings.

Purine anabolism creates therapeutic vulnerability in hepatocellular carcinoma through m 6 A-mediated epitranscriptomic regulation.

Purines are building blocks for the cellular genome, and excessive purine nucleotides are seen in tumors. However, how purine metabolism is dysregulated in tumors, and impacting tumorigenesis remains elusive. Transcriptomic and metabolomic analyses of purine biosynthesis and purine degradation pathways were performed in the tumor and associated nontumor liver tissues obtained from 62 patients with HCC, one of the most lethal cancers worldwide. We found that most genes in purine synthesis are upregulated, while genes in purine degradation are inhibited in HCC tumors. High purine anabolism is associated with unique somatic mutational signatures linked to patient prognosis. Mechanistically, we discover that increasing purine anabolism promotes epitranscriptomic dysregulation of DNA damage repairing (DDR) machinery through upregulating RNA N6-methyladenosine (m 6 A) modification. High purine anabolic HCC is sensitive to DDR-targeting agents but not to standard HCC treatments, correlating with the clinical outcomes in 5 independent HCC cohorts containing 724 patients. We further showed that high purine anabolism determines the sensitivity to DDR-targeting agents in 5 HCC cell lines in vitro and in vivo . Our results reveal a central role of purine anabolism in regulating DDR, which could be therapeutically exploited in HCC.

Abstract 3682: Glutamine addiction of c-MAF overexpressing cells drives dysregulation of purine metabolism

Abstract c-MAF, a bZIP transcription factor, is overexpressed as a consequence of t(14;16) translocation and represents a driver oncogene in this subtype. Clinically, patients with t(14;16) translocation constitute a high-risk group with adverse survival outcomes. Since transcription factors are not classical targets for the development of small molecule inhibitors, elucidating pathways downstream of c-MAF could present as potential therapeutic targets. Critically, gene expression profiling studies of c-MAF showed a class of recurrently deregulated metabolic genes. Hence, this warrants further studies into the underexplored role of metabolism and the deregulated metabolic pathways mediated by c-MAF. First, we confirmed that t(14;16) cells were dependent on c-MAF expression for cell viability. Abrogation of c-MAF deregulated sensitivity to glycolytic inhibitors and influenced metabolite readouts. Importantly, analysis of quantitative metabolic profile of c-MAF driven myeloma using metabolomics identified 41% (48/116) of metabolites to vary significantly. Classification of differential metabolites into metabolic pathways was performed using the software MetaboAnalyst 5.0 to elucidate a role of c-MAF in de novo purine metabolism. Despite an increased source of PRPP derived from the pentose phosphate pathway, we observed significant decrease in output of IMP (fold change=0.6, p=0.028). We postulate that this might result from lowered levels of some non-essential amino acids substrates needed by enzymes in the de novo purine biosynthesis pathway such as glutamine (fold change=0.08, p=0.155) and aspartate (fold change=0.3, p=0.022). Furthermore, GSEA from RNA-seq data performed by c-MAF loss-of-function studies identified ADCY1, ADCY5 and GALR3, suggesting a direct regulation of purine metabolic genes by c-MAF. Notably, to validate whether c-MAF displayed metabolic dependency on glutamine to drive purine biosynthesis, we performed nutrient starvation studies which showed glutamine dependency in c-MAF overexpressed cells. We also systematically knockdown glucose transporters (GLUT8 and GLUT4) or glutamine transporters (ASCT2 and SNAT1) and saw a greater dependency on glutamine transporters for cell viability. As a proof of concept, c-MAF overexpressed cells were more sensitive to purine depletion by LTX suggesting its dependency on purine metabolism. The downstream identification of biological association of c-MAF with cancer metabolism may emerge as a new therapeutic strategy for MM patients. Taken together, our study suggests that metabolic perturbations present as a likely key Achilles heel in MM, and could potentially shift the current treatment paradigm. Citation Format: Julia Sze Lynn Lim, Phyllis Shu Yun Chong, Wee-Joo Chng. Glutamine addiction of c-MAF overexpressing cells drives dysregulation of purine metabolism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3682.

GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

Metabolomic and transcriptomic response to imatinib treatment of gastrointestinal stromal tumour in xenograft-bearing mice

BackgroundAlthough imatinib is a well-established first-line drug for treating a vast majority of gastrointestinal stromal tumours (GIST), GISTs acquire secondary resistance during therapy. Multi-omics approaches provide an integrated perspective to empower the development of personalised therapies through a better understanding of functional biology underlying the disease and molecular-driven selection of the best-targeted individualised therapy. In this study, we applied integrative metabolomic and transcriptomic analyses to elucidate tumour biochemical processes affected by imatinib treatment. Materials and methodsA GIST xenograft mouse model was used in the study, including 10 mice treated with imatinib and 10 non-treated controls. Metabolites in tumour extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). RNA sequencing was also performed on the samples subset (n=6). ResultsMetabolomic analysis revealed 21 differentiating metabolites, whereas next-generation RNA sequencing data analysis resulted in 531 differentially expressed genes. Imatinib significantly changed the profile of metabolites associated mainly with purine and pyrimidine metabolism, butanoate metabolism, as well as alanine, aspartate, and glutamate metabolism. The related changes in transcriptomic profiles included genes involved in kinase activity and immune responses, as well as supported its impact on the purine biosynthesis pathway. ConclusionsOur multi-omics study confirmed previously known pathways involved in imatinib anticancer activity as well as correlated imatinib-relevant downregulation of expression of purine biosynthesis pathway genes with the reduction of respectful metabolites. Furthermore, considering the importance of the purine biosynthesis pathway for cancer proliferation, we identified a potentially novel mechanism for the anti-tumour activity of imatinib. Based on the results, we hypothesise metabolic modulations aiming at the reduction in purine and pyrimidine pool may ensure higher imatinib efficacy or re-sensitise imatinib-resistant tumours.

Transcriptomic and metabolomic analyses reveal the antifungal mechanism of the compound phenazine-1-carboxamide on Rhizoctonia solani AG1IA.

To explore the molecular mechanisms of the antifungal compound phenazine-1-carboxamide (PCN) inhibits Rhizoctonia solani and discover potential targets of action, we performed an integrated analysis of transcriptome and metabolome in R. solani mycelium by whether PCN treating or not. A total of 511 differentially expressed genes (DEGs) were identified between the PCN treatment and control groups. The fluorescence-based quantitative PCR (qPCR) got the accordant results of the gene expression trends for ten randomly selected DEGs. The Gene Ontology (GO) enrichment analysis revealed that fatty acid metabolic process, fatty acid oxidation, and lipid oxidation were among the most enriched in the biological process category, while integral component of membrane, plasma membrane, and extracellular region were among the most enriched in the cellular component category and oxidoreductase activity, cofactor binding, and coenzyme binding were among the most enriched in the molecular function category. KEGG enrichment analysis revealed the most prominently enriched metabolic pathways included ATP-binding cassette (ABC) transporters, nitrogen metabolism, aminobenzoate degradation. The DEGs related functions of cellular structures, cell membrane functions, cellular nutrition, vacuole-mitochondrion membrane contact site and ATPase activity, pH, anti-oxidation, were downregulated. A total of 466 differential metabolites were found between the PCN treatment and control groups after PCN treatment. KEGG enrichment found purine, arachidonic acid, and phenylpropanoid biosynthesis pathways were mainly affected. Further results proved PCN decreased the mycelial biomass and protein content of R. solani, and superoxide dismutase (SOD) activity reduced while peroxidase (POD) and cytochrome P450 activities increased. The molecule docking indicted that NADPH nitrite reductase, ATP-binding cassette transporter, alpha/beta hydrolase family domain-containing protein, and NADPH-cytochrome P450 reductase maybe the particular target of PCN. In conclusion, the mechanisms via which PCN inhibits R. solani AG1IA may be related to cell wall damage, cell membrane impairment, intracellular nutrient imbalance, disturbed antioxidant system, and altered intracellular pH, which laid foundation for the further new compound designing to improve antifungal efficacy.

TMET-35. ONE CARBON AND NUCLEOTIDE METABOLISM ARE NOVEL TARGETS IN PEDIATRIC BRAINSTEM TUMORS

Abstract Diffuse intrinsic pontine glioma (DIPG) are inoperable tumors of the brainstem with no cure. The median survival of children with DIPG is less than 2 years and 5-year overall survival is only 1%. Genomic studies have identified recurrent mutations in histone H3, ACVR and TP53 genes in DIPG. However, there are still no breakthrough drug targets for DIPG. To better understand the disease biology and find new drug targets for DIPG, we recently performed joint pathway analysis of DIPG metabolites (untargeted metabolomics) and differentially expressed genes (RNAseq) relative to normal human neural stem cells. We discovered that specific enzymes of the de novo purine biosynthesis pathway (DNPB), serine synthesis and the associated and one-carbon (1C) metabolism are highly active and the most upregulated in DIPG tumors and cell lines. Gain and loss of function experiments as well as pharmacological studies indicated a mechanistic link between DIPG mutations and DNPB pathway and the dependence of DIPG cells on specific DNPB and 1C metabolism enzymes for in vitro and in vivo growth. The 1C pathway is crucial for rapidly proliferating malignant cells as it senses and regulates cellular nutrient status by allocating and cycling 1C-groups between different acceptor compounds. It controls synthesis of nucleotides, amino acids, glycogen and phosphoglyceride precursors, S-adenosylmethionine (SAM), glutathione and NADPH. Thus, besides dispensing carbon atoms it also regulates cellular epigenetic and redox status. An important carbon donor is serine which is synthesized from glucose at 3-phosphoglycerate step in glycolysis. Untargeted as well as carbon and nitrogen tracing experiments indicated large folate pools in DIPG cells that can serve as a buffer to potentially blunt antifolate therapy. Experiments are underway to understand the importance of the folate cycle in DIPG and design novel and translatable combination therapies for DIPG.

Identification of genetic elements required for Listeria monocytogenes growth under limited nutrient conditions and virulence by a screening of transposon insertion library.

Listeria monocytogenes, the causative agent of listeriosis, displays a lifestyle ranging from saprophytes in the soil to pathogenic as a facultative intracellular parasite in host cells. In the current study, a random transposon (Tn) insertion library was constructed in L. monocytogenes strain F2365 and screened to identify genes and pathways affecting in vitro growth and fitness in minimal medium (MM) containing different single carbohydrate as the sole carbon source. About 2,000 Tn-mutants were screened for impaired growth in MM with one of the following carbon sources: glucose, fructose, mannose, mannitol, sucrose, glycerol, and glucose 6-phosphate (G6P). Impaired or abolished growth of L. monocytogenes was observed for twenty-one Tn-mutants with disruptions in genes encoding purine biosynthesis enzymes (purL, purC, purA, and purM), pyrimidine biosynthesis proteins (pyrE and pyrC), ATP synthase (atpI and atpD2), branched-chain fatty acids (BCFA) synthesis enzyme (bkdA1), a putative lipoprotein (LMOF2365_2387 described as LP2387), dUTPase family protein (dUTPase), and two hypothetical proteins. All Tn-mutants, except the atpD2 mutant, grew as efficiently as wild-type strain in a nutrient rich media. The virulence of twenty-one Tn-mutants was assessed in mice at 72 h following intravenous (IV) infection. The most attenuated mutants had Tn insertions in purA, hypothetical protein (LMOf2365_0064 described as HP64), bkdA1, dUTPase, LP2387, and atpD2, confirming the important role of these genes in pathogenesis. Six Tn-mutants were then tested for ability to replicate intracellularly in murine macrophage J774.1 cells. Significant intracellular growth defects were observed in two Tn-mutants with insertions in purA and HP64 genes, suggesting that an intact purine biosynthesis pathway is important for intracellular growth of L. monocytogens. These findings may not be fully generalized to all of L. monocytogenes strains due to their genetic diversity. In conclusion, Tn-mutagenesis identified that biosynthesis of purines, pyrimidines, ATP, and BCFA are important for L. monocytogens pathogenesis. Purine and pyrimidine auxotrophs play an important role in the pathogenicity in other bacterial pathogens, but our study also revealed new proteins essential for both growth in MM and L. monocytogenes strain F2365 virulence.

Riboswitch-mediated regulation of riboflavin biosynthesis genes in prokaryotes.

Prokaryotic organisms frequently use riboswitches to quantify intracellular metabolite concentration via high-affinity metabolite receptors. Riboswitches possess a metabolite-sensing system that controls gene regulation in a cis-acting fashion at the initiation of transcriptional/translational level by binding with a specific metabolite and controlling various biochemical pathways. Riboswitch binds with flavin mononucleotide (FMN), a phosphorylated form of riboflavin and controls gene expression involved in riboflavin biosynthesis and transport pathway. The first step of the riboflavin biosynthesis pathway is initiated by the conversion of guanine nucleotide triphosphate (GTP), which is an intermediate of the purine biosynthesis pathway. An alternative pentose phosphate pathway of riboflavin biosynthesis includes the enzymatic conversion of ribulose-5-phosphate into 3, 4 dihydroxy-2-butanone-4-phosphates by DHBP synthase. The product of ribAB interferes with both GTP cyclohydrolase II as well as DHBP synthase activities, which catalyze the cleavage of GTP and converts DHBP Ribu5P in the initial steps of both riboflavin biosynthesis branches. Riboswitches are located in the 5' untranslated region (5' UTR) of messenger RNAs and contain an aptamer domain (highly conserved in sequence) where metabolite binding leads to a conformational change in an aptamer domain, which modulate the regulation of gene expression located on bacterial mRNA. In this review, we focus on how riboswitch regulates the riboflavin biosynthesis pathway in Bacillus subtilis and Lactobacillus plantarum.

De Novo Purine Nucleotide Biosynthesis Pathway Is Required for Development and Pathogenicity in Magnaporthe oryzae.

Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.

The improved genome of the nematode Parapristionchus giblindavisi provides insights into lineage-specific gene family evolution.

Nematodes such as Caenorhabditis elegans and Pristionchus pacificus are extremely successful model organisms for comparative biology. Several studies have shown that phenotypic novelty but also conserved processes are controlled by taxon-restricted genes. To trace back the evolution of such new or rapidly evolving genes, a robust phylogenomic framework is indispensable. Here, we present an improved version of the genome of Parapristionchus giblindavisi which is the only known member of the sister group of Pristionchus. Relative to the previous short-read assembly, the new genome is based on long reads and displays higher levels of contiguity, completeness, and correctness. Specifically, the number of contigs dropped from over 7,303 to 735 resulting in an N50 increase from 112 to 791 kb. We made use of the new genome to revisit the evolution of multiple gene families. This revealed Pristionchus-specific expansions of several environmentally responsive gene families and a Pristionchus-specific loss of the de novo purine biosynthesis pathway. Focusing on the evolution of sulfatases and sulfotransferases, which control the mouth form plasticity in P. pacificus, reveals differences in copy number and genomic configurations between the genera Pristionchus and Parapristionchus. Altogether, this demonstrates the utility of the P. giblindavisi genome to date and polarizes lineage-specific patterns.

MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.