Analysis Of Intracellular Metabolites Research Articles

A40 THE ROLE OF HYPOXIA-INDUCIBLE FACTOR IN CELLULAR METABOLIC ADAPTATION UPON GIARDIA INFECTION

Abstract Background Recent findings have highlighted the integral role of oxygen tension in gastrointestinal (GI) homeostasis. During hypoxia when oxygen supply is limited, mammalian cells utilize the Hypoxia-Inducible Factor (HIF) complex to activate genes that promote cell survival and alter glucose metabolism. The GI system exists in a state of physiologic hypoxia, subject to further alterations by pathogens. Although tissue and blood parasites are known to influence tissue oxygen tension, little is known regarding the modulation of hypoxia and HIF by enteric parasites. Aims This project aims to characterize the effect of Giardia duodenalis, a common enteric protozoan that perturbs gastrointestinal function leading to diarrheal disease, on the cellular hypoxic response. We hypothesize that hypoxia-associated genes are activated upon Giardia infection, leading to an adaptive metabolic response with increased glycolytic flux to maintain cellular bioenergetic homeostasis. Methods Caco-2 colonic epithelial cells were infected with Giardia isolate GS/M (MOI 10) for 1.5 or 4.5 hours to capture early and peak HIF activation, under normoxic (21% O2) or hypoxic (1%O2, STEMCELL hypoxia incubator) conditions. RNA was extracted for RT-qPCR to assess transcriptional changes of known HIF-target genes. Intracellular metabolite analysis via liquid-chromatography mass-spectrometry (hydrophilic-interaction chromatography method) was performed to assess HIF-mediated metabolic changes in uninfected and Giardia-infected cells (MOI 10) exposed to the hypoxia mimetic DMOG or the HIF inhibitor PX-478. Results Under normoxic conditions, genes associated with compensatory cellular stress responses such as VEGFA, ANKRD37, GADD45A, and glycolysis-associated genes HK2, and LDHA are upregulated in Giardia-infected cells in a time-dependent manner (pampersand:003C0.05). Under hypoxic conditions, fewer HIF-target genes are upregulated (e.g., VEGFA, GADD45A, PGK1; pampersand:003C0.05), suggesting Giardia-infected cells behave similarly to uninfected cells with constant HIF activation. Analysis of the Caco-2 intracellular metabolome indicates HIF-dependent changes in amino acid (e.g., glycine, threonine) and nucleic acid (e.g., guanosine, inosine, uracil) metabolism. DMOG treatment altered the production of glycolytic intermediates (e.g., DHAP, PEP), confirming the promotion of glycolytic flux by HIF. Conclusions Both cell-stress-related and glucose-metabolism HIF target genes are activated upon Giardia infection. As well, some metabolic changes are dependent on HIF activation. These novel findings indicate Giardia promotes a hypoxic state in human intestinal cells, highlighting a metabolic cell rescue mechanism in response to enteropathogens. Furthermore, understanding the role of hypoxia in enteric parasitic infections will shed light on the potential of HIF as a therapeutic target. Funding Agencies Natural Sciences and Engineering Research Council of Canada (NSERC)

Abstract 3104: Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1

Abstract Introduction. Activating the immune system against cancer is becoming an increasingly effective therapy option that can result in dramatic and durable responses in several cancer types. One approach to achieve the reactivation of endogenous antitumor T cells is by blocking PD-1/PD-L1 immune checkpoints expressed on T cells and other leukocytes. However, only limited cancer patients (15-25%) respond to anti-PD-1/PD-L1 immunotherapy. One of the most pressing current clinical challenges is to convert nonresponsive, “cold” tumors to responsive, “hot” tumors. Interestingly, after DNA-damaging chemotherapy, the immune environment may be changed from “cold” tumors to “hot” tumors by increasing the tumor mutation burden and the generation of neoantigens on the surface of cancer cells. Therefore, we surmised that a molecule capable of inducing promutagenic DNA and block PD-1/PD-L1 could not only induce neoantigens but also synergistically enhance immune response against the targeted tumor. Using an approach developed in our laboratory termed the “combi-targeting” strategy, we designed and synthesized a series of “combi-molecules” programmed to generate the promutagenic species and a small molecule capable of blocking PD-1/PD-L1. Material and methods. Melanoma cell line B16-F10 was used to determine IC50 of the new molecules with SRB assay. Homogenous time-resolved fluorescence (HTRF) binding assay was used to determine the IC50 inhibition of PD-1/PD-L1. Drug metabolism in extracted cells was measured by LC-MS. Results. We discovered a structure activity relationship of the combi-molecules based on the substitution of the side chain of the alkylating agent and the PD-1/PD-L1 scaffold. By altering the scaffolds of the combi-molecules from sulfonamides to biphenyl derivatives, we optimized binding to PD-1/PD-L1 from millimolar to micromolar levels. In vitro growth inhibitory analysis showed that the combi-molecules with biphenyl scaffold were 24-fold more potent in B16-F10. Importantly, analysis of intracellular metabolites of the combi-molecules revealed three main metabolites that can only result from the release of the shortlived promutagenic species. Conclusions. The biphenyl scaffold is optimal for maintaining strong PD-1/PD-L1 binding potency and enough potent to contribute to cell death. Hydrolytic cleavage of the alkylating agent is an indirect evidence of the formation of the alkylating species required to induce promutagenic lesions. Citation Format: Ana Belen Fraga Timiraos, Caterina Facchin, Nadia Babaa, Anne-Laure Larroque-Lombard, Bertrand Jean-Claude. Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1 [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 3104.

Metabolic engineering of B. subtilis 168 for increased precursor supply and poly-γ-glutamic acid production

Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer produced by several Bacillus species. To improve γ-PGA synthesis, metabolic engineering of the production host B. subtilis poses great potential and is facilitated by the convenient genetical amenability of the organism. In this study, a 3.7-fold increase in γ-PGA production using a bdhA, alsSD, pta, yvmC, and cypX deletion mutant with blocked by-product synthesis pathways was obtained. A detailed analysis of intracellular metabolites for reference strains and the γ-PGA-producing deletion strain identified the accumulation of pyruvate and acetyl-CoA in deletion mutants, highlighting the citrate synthase activity as an important metabolic engineering target for further metabolic flux optimization towards γ-PGA synthesis. An in-depth analysis of growth and γ-PGA production with on-line measurement techniques revealed significant variations across cultivations with deletion mutants that are likely caused by culture acidification due to pyruvate accumulation. Despite the observed acidification, the by-product deletion mutants outperformed the reference strains independent of the promoter controlling the PGA synthetase expression. The constructed deletion strains exhibit high γ-PGA production in minimal medium with glucose as sole carbon source as well as in modified Medium E reaching γ-PGA concentrations of 0.57 gL-1 and 14.46 gL-1, respectively. The results presented in this work broaden the understanding of the microbial metabolism during γ-PGA production and will be useful to guide future metabolic engineering for improved γ-PGA production.

VAMS-Based Blood Capillary Sampling for Mass Spectrometry-Based Human Metabolomics Studies

Volumetric absorptive microsampling (VAMS) is a recently developed sample collection method that enables single-drop blood collection in a minimally invasive manner. Blood biomolecules can then be extracted and processed for analysis using several analytical platforms. The integration of VAMS with conventional mass spectrometry (MS)-based metabolomics approaches is an attractive solution for human studies representing a less-invasive procedure compared to phlebotomy with the additional potential for remote sample collection. However, as we recently demonstrated, VAMS samples require long-term storage at -80 °C. This study investigated the stability of VAMS samples during short-term storage and compared the metabolome obtained from capillary blood collected from the fingertip to those of plasma and venous blood from 22 healthy volunteers. Our results suggest that the blood metabolome collected by VAMS samples is stable at room temperature only for up to 6 h requiring subsequent storage at -80 °C to avoid significant changes in the metabolome. We also demonstrated that capillary blood provides better coverage of the metabolome compared to plasma enabling the analysis of several intracellular metabolites presented in red blood cells. Finally, this work demonstrates that with the appropriate pre-analytical protocol capillary blood can be successfully used for untargeted metabolomics studies.

Quantitation of methotrexate polyglutamates in human whole blood, erythrocytes and leukocytes collected via venepuncture and volumetric absorptive micro-sampling: a green LC–MS/MS-based method

Low-dose methotrexate (MTX) plays a key role in treatment of rheumatoid arthritis. However, not all patients respond satisfactorily, and no therapeutic drug monitoring has been implemented in clinical practice, despite the fact that MTX therapy has now been available for decades. Analysis of individual intracellular MTX metabolites among rheumatoid arthritis (RA) patients is hampered by the low intracellular concentrations of MTX-PGs which require a highly sensitive method to quantify. Here, we present a rapid and highly sensitive LC (HILIC) MS/MS method with LLOQ 0.1 nM, 0.8 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7 respectively. Over a linear range of 0.1–100 nM, 0.8–100 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7, respectively, the inter- and intra- accuracy and precision were within 15% of the nominal value for all MTX metabolites. The presented assay was used to assess and compare MTX metabolite concentrations extracted from four different matrices: red blood cells, plasma, peripheral blood mononuclear cells, and whole blood that have been collected either using traditional venepuncture or volumetric absorptive micro-sampling (VAMS) sampling techniques. The presented method not only improves analyte coverage and sensitivity as compared to other published methods; it also improves the greenness.Graphical abstract

The Role of Acetate Kinase in the Human Parasite Entamoeba histolytica.

The human parasite Entamoeba histolytica, which causes approximately 100 million cases of amoebic dysentery each year, relies on glycolysis as the major source of ATP production from glucose as it lacks a citric acid cycle and oxidative phosphorylation. Ethanol and acetate, the two major glycolytic end products for E. histolytica, are produced at a ratio of 2:1 under anaerobic conditions, creating an imbalance between NADH production and utilization. In this study we investigated the role of acetate kinase (ACK) in acetate production during glycolysis in E. histolytica metabolism. Analysis of intracellular and extracellular metabolites demonstrated that acetate levels were unaffected in an ACK RNAi cell line, but acetyl-CoA levels and the NAD+/NADH ratio were significantly elevated. Moreover, we demonstrated that glyceraldehyde 3-phosphate dehydrogenase catalyzes the ACK-dependent conversion of acetaldehyde to acetyl phosphate in E. histolytica. We propose that ACK is not a major contributor to acetate production, but instead provides a mechanism for maintaining the NAD+/NADH balance during ethanol production in the extended glycolytic pathway.

Screening of Carbofuran-Degrading Bacteria Chryseobacterium sp. BSC2-3 and Unveiling the Change in Metabolome during Carbofuran Degradation.

Carbofuran is one of the most commonly used N-methylcarbamate-based pesticides and is excellent for controlling pests; however, carbofuran also causes soil and water pollution. Although various studies have been conducted on the bioremediation of pesticide-contaminated soil, the changes occurring in the metabolome during the bioremediation of carbofuran are not fully understood. In this study, the intracellular and extracellular metabolites of the Chryseobacterium sp. BSC2-3 strain were analysed during carbofuran degradation by using a liquid chromatography–mass spectrometry-based metabolomics approach. We found that the BSC2-3 strain extracellularly transformed carbofuran into 3-hydroxycarbofuran. Intracellular metabolite analysis revealed that carbofuran mainly affected aminobenzoate degradation, ubiquinone and terpenoid-quinone biosynthesis, and arginine and proline metabolism. Carbofuran especially affected the metabolic pathway for the degradation of naphthalene and aminobenzoate. Metabolomics additionally revealed that the strain produces disease resistance inducers and plant growth regulators. We also identified the genes involved in the production of indole-3-acetic acid, which is one of the most active auxins. Overall, we identified the metabolic changes induced in carbofuran-degrading bacteria and the genes predicted to be responsible for the degradation of carbofuran.

Analysing intracellular isoprenoid metabolites in diverse prokaryotic and eukaryotic microbes.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

A Minority Population of Non-dye-decolorizing Bacillus subtilis enhances the Azo Dye-decolorizing Activity of Enterococcus faecalis.

Microbes live in communities in biological wastewater treatment plants and in the intestines. However, limited information is currently available on the mechanisms by which minority bacterial populations assist other bacteria besides syntrophic relationships as well as on the microbial food web. Therefore, the present study investigated the effects of non-dye-decolorizing Bacillus subtilis strain S4ga at population levels ranging between 0.04 and 4% on the activity of dye-decolorizing Enterococcus faecalis strain T6a1 using a dye decolorization assay. The results obtained revealed that the minority population of B. subtilis S4ga enhanced the dye-decolorizing activity of E. faecalis T6a1, resulting in a shorter lag time and longer active time of dye decolorization. These effects were related to redox potential values rather than O2 concentrations. Comparisons of the extracellular metabolites in individual incubations of E. faecalis T6a1 and B. subtilis S4ga and a co-incubation suggested a mutual relationship through the cross-feeding of specific amino acids (tyrosine, methionine, tryptophan, phenylalanine, valine, and leucine from B. subtilis S4ga to E. faecalis T6a1; glutamine, histidine, aspartic acid, and proline from E. faecalis T6a1 to B. subtilis S4ga). An ana­lysis of intracellular primary metabolites indicated that the arginine deiminase (ADI) pathway, an ATP-producing energy-generating process, was more strongly activated in co-incubated E. faecalis T6a1 than in E. faecalis T6a1 incubated alone. These results suggest that a co-incubation with B. subtilis S4ga promoted ATP production by E. faecalis T6a1 cells and enhanced its dye-decolorizing activity.

Resveratrol production of a recombinant Scheffersomyces stipitis strain from molasses

Resveratrol is a plant-derived aromatic compound with beneficial properties and it is required to develop a resveratrol production process from inexpensive biomass feedstocks. Here, we investigated the potential of Scheffersomyces stipitis, a non-conventional yeast with the capacity to utilize a wide range of sugars, to produce resveratrol from molasses, which is a by-product of sugar refineries. The S. stipitis strain metabolically engineered for resveratrol production produced resveratrol from 60 g/L mixed sugar (sucrose, glucose, and fructose), while its resveratrol titer decreased as the proportions of glucose and fructose increased. Sucrose consumption of the S. stipitis strain was clearly suppressed by the coexistence of glucose, fructose, and even ethanol. Quantitative analysis of intracellular metabolites involved in resveratrol biosynthesis using capillary electrophoresis time-of-flight mass spectrometry revealed that the composition of these sugars has a significant effect on the intracellular accumulation of glycolytic metabolites and AMP, which is an important factor involved in some cellular metabolic responses. Furthermore, the S. stipitis strain produced 1076 ± 167 mg/L of resveratrol in the fermentation with commercial sugarcane molasses (120 g/L of total sugars) as the substrate. To our knowledge, this is the first report on carbon catabolite repression in S. stipitis caused by the coexistence of sucrose, glucose, and fructose and resveratrol production from molasses. These results indicate great potential of the cost-effective resveratrol production process from molasses substrates using recombinant S. stipitis.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 μM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

SNF1β-Modulated Glucose Uptake and the Balance between Polyunsaturated Fatty Acids and Carbohydrates in Mortierella alpina.

A sucrose nonfermenting protein kinase 1 (SNF1) complex is an important metabolic regulator in fungi that is critical to cell metabolism and stress response. In this study, the role of an SNF1 β-subunit in the oleaginous fungus Mortierella alpina (MaSip2) was investigated. The MaSip2 contained a glycogen-binding domain and a conserved SNF1-complex interaction region; its transcriptional level during lipogenesis shared high consistency with a previously reported SNF1 γ-subunit (MaSnf4). Overexpression of MaSip2 in M. alpina significantly promoted glucose uptake and resulted in 34.1% increased total biomass, leading to 44.8% increased arachidonic acid yield after 7 day fermentation. MaSip2 also regulated the balance between polyunsaturated fatty acids and carbohydrates in M. alpina. Intracellular metabolite analysis revealed increased carbohydrate-related metabolite accumulation in MaSip2 overexpression strains. On the contrary, knockdown of MaSip2 increased the total fatty acid unsaturation degree, especially under low-temperature conditions. This research improved our knowledge of SNF1 complex in M. alpina and provided a target gene for enhancing glucose utilization and modulating fatty acid composition for better application of oleaginous fungi.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

BackgroundSugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica.ResultsY. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production.ConclusionsWe demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

Deciphering metabolic alterations in algae cultivated in spent media as means for enhancing algal biorefinery sustainability

The recycling of unfiltered spent media during cultivation of Chlorella vulgaris was studied using metabolomics in an effort to enhance water and nutrient sustainability and reduce operating costs in algal biorefineries. Cultivation in spent media resulted in reduced biomass and lipid productivity by 14% and 19%, respectively, compared to fresh media. The decrease was related to a detected lower nutrient uptake. Nevertheless, carbohydrate content (28% of dry cell weight) and α-linolenic acid content (27 % of fatty acids) were higher in spent media cultures than in fresh media. Metabolomics analysis of intracellular metabolites revealed downregulation of nitrogen assimilation, tricarboxylic acid cycle, structural lipids, and energy metabolism, but upregulation of stress mitigation and carbohydrate synthesis. No growth was supported by spent media during a second cultivation cycle and was likely due to the identified extracellular accumulation of humic acid and free fatty acids that acted as growth auto-inhibitors.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2

BackgroundPamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2.ResultsSupplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L−1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type.ConclusionsOur findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death.

Metabolic reprogramming within cancer cells has been recognized as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is the one of factors behind discernible hallmarks such as drug resistance, relapse of the tumor and the formation of secondary tumors. In this paper, cell-based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in a hypotonic buffer and the whole cell lysate was purified by a novel and specifically designed metabolite (~ 100 to 1000Da) fractionation system called vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique. Cleaved PARP 1 in MCF-7 cells treated with DOX was observed in the present study. Concomitantly, data showed the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, we reported the first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that are potentially linked to the mechanism of cell death in MCF-7 cells. This paper reported novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells. Finally, these findings validate a proof of concept on the applications of novel VTGE assisted purification and analysis of intracellular metabolites from various cell culture models.

A possible influence of extracellular polysaccharides on the analysis of intracellular metabolites from Trichoderma harzianum grown under carbon-limited conditions

Intracellular metabolites were evaluated during the continuous growth of Trichoderma harzianum P49P11 under carbon-limited conditions. Four different conditions in duplicate were investigated (10 and 20 g/L of glucose, 5.26/5.26 g/L of fructose/glucose and 10 g/L of sucrose in the feed). Differences in the values of some specific concentrations of intracellular metabolites were observed at steady-state for the duplicates. The presence of extracellular polysaccharide was confirmed in the supernatant of all conditions based on FT-IR and proton NMR. Fragments of polysaccharides from the cell wall could be released due to the shear stress and since the cells can consume them under carbon-limited conditions, this could create an unpredictable carbon flow rate into the cells. According to the values of the metabolite concentrations, it was considered that the consumption of those fragments was interfering with the analysis.

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A.kawachii cexA gene (AkcexA) into A.oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A.oryzae to a level comparable to that of A.kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A.oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A.oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

Metabolic reprogramming related to whole-chromosome instability in models for H\xfcrthle cell carcinoma

Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2–7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG.