scholarly journals Non-random organization of flux control mechanisms in yeast central metabolic pathways

2021 ◽  
Author(s):  
Rosemary Yu ◽  
Egor Vorontsov ◽  
Carina Sihlbom ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Metabolic Network ◽  
Metabolic Pathways ◽  
Evolutionary History ◽  
Metabolic Flux ◽  
Metabolic Modeling ◽  
Control Mechanisms ◽  
Flux Control ◽  
Regulation Of Metabolism

Metabolic flux can be regulated by a variety of different mechanisms, but the organization of these mechanisms within the metabolic network has remained unknown. Here we test the hypothesis that flux control mechanisms are not distributed randomly in the metabolic network, but rather organized according to pathway. Combining proteomics, phosphoproteomics, and metabolic modeling, we report the largest collection of flux-enzyme-phosphoenzyme relationships to date in Saccharomyces cerevisiae. In support of the hypothesis, we show that (i) amino acid metabolic pathways are predominantly regulated by enzyme abundance stemming from transcriptional regulation; (ii) upper glycolysis and associated pathways, by inactivating enzyme phosphorylation; (iii) lower glycolysis and associated pathways, by activating enzyme phosphorylation; and (iv) glycolipid/glycophospholipid pathways, by a combination of enzyme phosphorylation and metabolic compartmentalization. We delineate the evolutionary history for the observed organization of flux control mechanisms in yeast central metabolic pathways, furthering our understanding of the regulation of metabolism and its evolution.

Pyridoxal and α-ketoglutarate independently improve function of Saccharomyces cerevisiae Thi5 in the metabolic network of Salmonella enterica

2021 ◽  
Author(s):  
Michael D. Paxhia ◽  
Diana M. Downs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Network ◽  
Legionella Pneumophila ◽  
Salmonella Enterica ◽  
Metabolic Flux ◽  
Genetic Suppressors ◽  
Metabolite Pool ◽  
Improved Function ◽  
Dependent Pathway

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica . Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report a strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica . Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica . IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contribute to their functional differences in a Salmonella enterica host.

Drug Target Selection for Trypanosoma cruzi Metabolism by Metabolic Control Analysis and Kinetic Modeling

2019 ◽  
Vol 26 (36) ◽  
pp. 6652-6671 ◽  
Author(s):  
Emma Saavedra ◽  
Zabdi González-Chávez ◽  
Rafael Moreno-Sánchez ◽  
Paul A.M. Michels
Keyword(s):  
Trypanosoma Cruzi ◽  
Metabolic Control ◽  
Kinetic Modeling ◽  
Metabolic Pathways ◽  
Drug Target ◽  
Metabolic Modeling ◽  
Metabolic Control Analysis ◽  
Control Analysis ◽  
Control Coefficient ◽  
Flux Control

In the search for therapeutic targets in the intermediary metabolism of trypanosomatids the gene essentiality criterion as determined by using knock-out and knock-down genetic strategies is commonly applied. As most of the evaluated enzymes/transporters have turned out to be essential for parasite survival, additional criteria and approaches are clearly required for suitable drug target prioritization. The fundamentals of Metabolic Control Analysis (MCA; an approach in the study of control and regulation of metabolism) and kinetic modeling of metabolic pathways (a bottom-up systems biology approach) allow quantification of the degree of control that each enzyme exerts on the pathway flux (flux control coefficient) and metabolic intermediate concentrations (concentration control coefficient). MCA studies have demonstrated that metabolic pathways usually have two or three enzymes with the highest control of flux; their inhibition has more negative effects on the pathway function than inhibition of enzymes exerting low flux control. Therefore, the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention. In this review, the fundamentals of MCA as well as experimental strategies to determine the flux control coefficients and metabolic modeling are analyzed. MCA and kinetic modeling have been applied to trypanothione metabolism in Trypanosoma cruzi and the model predictions subsequently validated in vivo. The results showed that three out of ten enzyme reactions analyzed in the T. cruzi anti-oxidant metabolism were the most controlling enzymes. Hence, MCA and metabolic modeling allow a further step in target prioritization for drug development against trypanosomatids and other parasites.

scholarly journals Metabolic Flux Hierarchy Prioritizes the Entner-Doudoroff Pathway for Carbohydrate Co-Utilization inPseudomonas protegensPf-5

2018 ◽  
Author(s):  
Rebecca A. Wilkes ◽  
Caroll M. Mendonca ◽  
Ludmilla Aristilde
Keyword(s):  
Metabolic Network ◽  
Carbohydrate Metabolism ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Gene Annotation ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Environmental Matrices ◽  
Fructose 1,6 Bisphosphate ◽  
Kinase Phosphorylation

ABSTRACTThe genetic characterization ofPseudomonas protegensPf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here we employed13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. Similar to otherPseudomonasspecies,P. protegensPf-5 relied primarily on the Entner-Doudoroff (ED) pathway to connect initial glucose catabolism to downstream metabolic pathways. Flux quantitation determined that, in lieu of the direct phosphorylation of glucose by glucose kinase, phosphorylation of oxidized products of glucose (gluconate and 2-ketogluconate) towards the ED pathway accounted for over 90% of consumed glucose and greater than 35% of consumed glucose was secreted as gluconate and 2-ketogluconate. Consistent with the lack of annotated pathways for the initial catabolism of pentoses and galactose inP. protegensPf-5, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of the hexoses was evident but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism toward fructose 6-phosphate, metabolite labeling patterns revealed that mannose-derived carbons specifically entered central carbon metabolism via fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were found to cycle backward through the upper Emden-Meyerhof-Parnas pathway to feed into the ED pathway. Therefore, the operational metabolic network for processing carbohydrates inP. protegensPf-5 prioritizes flux through the ED pathway to channel carbons to downstream metabolic pathways.IMPORTANCESpecies of thePseudomonasgenus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here we investigated the metabolic network for carbohydrate metabolism inP. protegensPf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through the network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the gene annotation of two different pathways in the genome. Elucidation of the constitutive metabolic network inP. protegensis important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untappedPseudomonasspecies.

Biological Processing of Pea Grain and Secondary Starch Raw Materials to Produce Food and Feed Protein Concentrates

2020 ◽  
Vol 36 (4) ◽  
pp. 49-58
Author(s):  
V.V. Kolpakova ◽  
R.V. Ulanova ◽  
L.V. Chumikina ◽  
V.V. Bessonov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Mass Fraction ◽  
Geotrichum Candidum ◽  
Protein Concentrate ◽  
Protein Concentrates ◽  
Pea Flour ◽  
Nitrogenous Substances

The goal of the study was to develop a biotechnological process for the production of protein concentrates via bioconversion of pea flour and whey, a secondary product of starch manufacture. Standard and special methods were used to analyze the chemical and biochemical composition of protein concentrates (amino acid, carbohydrate, and fractional) of flour, whey and protein concentrates. It was established that pea flour contains 52.28-57.05% water-soluble nitrogenous substances, 23.04-25.50% salt-soluble, 2.94-4.69% alcohol-soluble compounds, 0-0.61% of soluble glutenine, 6.67-10.40% alkali-soluble glutenine and 5.96-10.86% insoluble sclerotic substances. A mathematical model and optimal parameters of the enzymatic extraction of pea protein with a yield of 65-70% were developed. Ultrasonic exposure increased the yield of nitrogenous substances by 23.16 ± 0.69%, compared with the control without ultrasound. The protein concentrate had a mass fraction of nitrogenous substances of 72.48 ± 0.41% (Nx6.25) and a complete amino acid composition. The microbial conversion by the Saccharomyces cerevisiae 121 and Geotrichum candidum 977 cultures of starch whey which remained after protein precipitation allowed us to obtain feed concentrates from biomass and culture liquid with a protein mass fraction of 61.68-70.48% (Nx6.25). Protein concentrates positively affected the vital signs of rats and their excretory products. A technological scheme was developed to test the complex pea grain and starch whey processing under pilot conditions. pea, protein concentrate, extracts, whey, bioconversion, Geotrichum candidum, Saccharomyces cerevisiae, chemical composition, amino acid composition

scholarly journals Kog1/Raptor mediates metabolic rewiring during nutrient limitation by controlling SNF1/AMPK activity

2021 ◽  
Vol 7 (16) ◽  
pp. eabe5544
Author(s):  
Zeenat Rashida ◽  
Rajalakshmi Srinivasan ◽  
Meghana Cyanam ◽  
Sunil Laxman
Keyword(s):  
Amino Acid ◽  
Nutrient Limitation ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Carbon Allocation ◽  
Carbon Utilization ◽  
Acid Biosynthesis ◽  
Control Growth ◽  
Ampk Activity ◽  
Delayed Growth

In changing environments, cells modulate resource budgeting through distinct metabolic routes to control growth. Accordingly, the TORC1 and SNF1/AMPK pathways operate contrastingly in nutrient replete or limited environments to maintain homeostasis. The functions of TORC1 under glucose and amino acid limitation are relatively unknown. We identified a modified form of the yeast TORC1 component Kog1/Raptor, which exhibits delayed growth exclusively during glucose and amino acid limitations. Using this, we found a necessary function for Kog1 in these conditions where TORC1 kinase activity is undetectable. Metabolic flux and transcriptome analysis revealed that Kog1 controls SNF1-dependent carbon flux apportioning between glutamate/amino acid biosynthesis and gluconeogenesis. Kog1 regulates SNF1/AMPK activity and outputs and mediates a rapamycin-independent activation of the SNF1 targets Mig1 and Cat8. This enables effective glucose derepression, gluconeogenesis activation, and carbon allocation through different pathways. Therefore, Kog1 centrally regulates metabolic homeostasis and carbon utilization during nutrient limitation by managing SNF1 activity.

Identification of a Functional Domain Within the Essential Core of Histone H3 That Is Required for Telomeric and HM Silencing in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 447-452 ◽  
Author(s):  
Jeffrey S Thompson ◽  
Marilyn L Snow ◽  
Summer Giles ◽  
Leslie E McPherson ◽  
Michael Grunstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Histone H3 ◽  
Single Amino Acid ◽  
Functional Domain ◽  
Partial Loss ◽  
Histone Fold ◽  
Gal1 Promoter ◽  
Substitution Mutations

Abstract Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the α1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.

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