scholarly journals Optimizing a High Performing Multiplex-CRISPRi P. putida strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

2021 ◽  
Author(s):  
Jeffrey J Czajka ◽  
Deepanwita Banerjee ◽  
Thomas T Eng ◽  
Javier Menasalvas ◽  
Chunsheng Yan ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Fold Increase ◽  
Tca Cycle ◽  
Strain Engineering ◽  
Batch Cultivation ◽  
Cell Factory ◽  
Flux Analysis ◽  
Original Strain ◽  
Microbial Cell Factory ◽  
Metabolite Level

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

TAMI-80. CELLULAR METABOLISM IN DIFFUSE INTRINSIC PONTINE GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi215-vi215
Author(s):  
Omkar Ijare ◽  
Jeanne Manalo ◽  
Martyn Sharpe ◽  
David Baskin ◽  
Kumar Pichumani
Keyword(s):  
Metabolic Flux ◽  
Treatment Strategies ◽  
Cellular Metabolism ◽  
Tca Cycle ◽  
Pediatric Brain Tumors ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Glutamine Metabolism ◽  
Flux Analysis ◽  
Pontine Glioma ◽  
Carboxylation Pathway

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), treated with 11.0 mM [U-13C]glucose or 4.0 mM glutamine in DMEM (supplemented with 10% FBS). After 24 h, cells were harvested for NMR/GC-MS analysis. The 13C-isotopomer analysis revealed that SF8628 cells produced 25.26 ± 10.63% acetyl-CoA from [U-13C]glucose which is ~3.7 times higher than that produced from GBM cells (6.83 ± 0.76%; our previous work), suggesting that DIPGs are metabolically very active. [U-13C]glutamine metabolism showed that DIPG cells also have an active TCA cycle metabolism (citrate M+4; 40.07 ± 1.06%) and moderately active reductive carboxylation pathway (citrate M+5; 10.59 ± 1.13%). Inhibition of both glycolytic and glutaminolysis pathways will be valuable in developing treatment strategies for DIPGs and these studies are in progress.

scholarly journals Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

2006 ◽  
Vol 73 (3) ◽  
pp. 718-729 ◽  
Author(s):  
Yinjie J. Tang ◽  
Judy S. Hwang ◽  
David E. Wemmer ◽  
Jay D. Keasling
Keyword(s):  
Metabolic Pathway ◽  
Uptake Rate ◽  
Shake Flask ◽  
Metabolic Flux ◽  
Environmental Changes ◽  
Shewanella Oneidensis ◽  
Tca Cycle ◽  
Shake Flask Culture ◽  
Flux Analysis ◽  
Lactate Uptake

ABSTRACT The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

scholarly journals Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

2014 ◽  
Vol 197 (5) ◽  
pp. 943-950 ◽  
Author(s):  
Le You ◽  
Lian He ◽  
Yinjie J. Tang
Keyword(s):  
Metabolic Flux ◽  
Electron Flow ◽  
Calvin Cycle ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Succinic Semialdehyde ◽  
Succinic Semialdehyde Dehydrogenase ◽  
Content Type ◽  
Semialdehyde Dehydrogenase ◽  
Pcc 6803

This study investigated metabolic responses inSynechocystissp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O2evolution and ATP/NADPH generation by linear electron flow. Based on13C-metabolic flux analysis (13C-MFA) and RNA sequencing, we have found thatSynechocystissp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO2and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.

scholarly journals Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Khushboo Borah ◽  
Tom A. Mendum ◽  
Nathaniel D. Hawkins ◽  
Jane L. Ward ◽  
Michael H. Beale ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Glyoxylate Shunt ◽  
Citrate Cycle ◽  
Metabolic Fluxes ◽  
The Tca Cycle ◽  
Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

scholarly journals Long Noncoding RNA UCA1 Promotes Glutamine-Driven Anaplerosis of Bladder Cancer by Interacting With hnRNP I/L to Upregulate GPT2 Expression

2020 ◽  
Author(s):  
Hua Zhao ◽  
Wenjing Wu ◽  
Xu Li ◽  
Wei Chen
Keyword(s):  
Bladder Cancer ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Metabolic Flux ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Tca Cycle ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Flux Analysis

Abstract Background: Glutamine-driven anaplerosis maintains the tricarboxylic acid (TCA) cycle by replenishing its carbon source of intermediates with the glutamine-derived carbons in cancer cells. Long noncoding RNA urothelial cancer associated 1 (UCA1), initially identified in bladder cancer, is associated with multiple cellular processes, including metabolic reprogramming. However, its characteristics in the anaplerosis context of bladder cancer (BLCA) remains elusive. Methods: The mechanism of UCA1 bound to and facilitated the combination of hnRNP I/L to the promoter of GPT2 gene was investigated by RNA pulldown, qRT-PCR, western blot, dual luciferase reporter assays, immunohistochemical staining, chromatin immunoprecipitation and chromatin isolation by RNA purification. Metabolomics analysis and metabolic flux analysis were conducted to assess the effects of UCA1, hnRNP I/L, and GPT2 on metabolic reprogramming of BLCA.Results: We identified UCA1 as a binding partner of heterogeneous nuclear ribonucleoproteins (hnRNPs) I and L, RNA-binding proteins with no previously known role in metabolic reprogramming. UCA1 and hnRNP I/L profoundly affected glycolysis, TCA cycle, glutaminolysis, and viability of BLCA cells. Importantly, UCA1 specifically bound to and facilitated the combination of hnRNP I/L to the promoter of glutamic pyruvate transaminase 2 (GPT2) gene, resulting in upregulated expression of GPT2 and enhanced glutamine-derived carbons in the TCA cycle. We also systematically confirmed the influence of UCA1, hnRNP I/L, and GPT2 on metabolism and proliferation via glutamine-driven anaplerosis in BLCA cells. Conclusions: Our study reveals the critical mechanism by which UCA1 forms a functional UCA1-hnRNP I/L complex that upregulates GPT2 expression to promote glutamine-driven TCA cycle anaplerosis, providing novel evidence that lncRNA regulates metabolic reprogramming in tumor cells.

scholarly journals Quantitative analysis of the physiological contributions of glucose to the TCA cycle

2019 ◽  
Author(s):  
Shiyu Liu ◽  
Ziwei Dai ◽  
Daniel E. Cooper ◽  
David G. Kirsch ◽  
Jason W. Locasale
Keyword(s):  
Quantitative Analysis ◽  
Metabolic Flux ◽  
Isotope Labeling ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Experimental Conditions ◽  
The Tca Cycle ◽  
Metabolic Physiology

ABSTRACTThe carbon source for catabolism in vivo is a fundamental question in metabolic physiology. Limited by data and rigorous mathematical analysis, controversy exists over the nutritional sources for carbon in the tricarboxylic acid (TCA) cycle under physiological settings. Using isotope-labeling data in vivo across several experimental conditions, we construct multiple models of central carbon metabolism and develop methods based on metabolic flux analysis (MFA) to solve for the preferences of glucose, lactate, and other nutrients used in the TCA cycle across many tissues. We show that in nearly all circumstances, glucose contributes more than lactate as a nutrient source for the TCA cycle. This conclusion is verified in different animal strains from different studies, different administrations of 13C glucose, and is extended to multiple tissue types. Thus, this quantitative analysis of organismal metabolism defines the relative contributions of nutrient fluxes in physiology, provides a resource for analysis of in vivo isotope tracing data, and concludes that glucose is the major nutrient used for catabolism in mammals.

scholarly journals Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

2010 ◽  
Vol 76 (19) ◽  
pp. 6529-6540 ◽  
Author(s):  
Vasiliy A. Portnoy ◽  
David A. Scott ◽  
Nathan E. Lewis ◽  
Yekaterina Tarasova ◽  
Andrei L. Osterman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Metabolic Flux ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Physiological Characterization ◽  
Metabolic Flux Distribution ◽  
K 12

ABSTRACT The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of d-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.

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