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.

scholarly journals Methionine coordinates a hierarchically organized anabolic program enabling proliferation

2018 ◽  
Vol 29 (26) ◽  
pp. 3183-3200 ◽  
Author(s):  
Adhish S. Walvekar ◽  
Rajalakshmi Srinivasan ◽  
Ritu Gupta ◽  
Sunil Laxman
Keyword(s):  
Amino Acid ◽  
Metabolic Flux ◽  
Response Regulator ◽  
Pentose Phosphate ◽  
Dependent Manner ◽  
Comparative Transcriptome ◽  
Starvation Stress ◽  
Acid Biosynthesis ◽  
Underlying Basis ◽  
Rate Limiting

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here we construct the organization of this methionine-mediated anabolic program using yeast. Combining comparative transcriptome analysis and biochemical and metabolic flux-based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH)-dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP)-dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine-mediated anabolic program to be sustained, cells co-opt a “starvation stress response” regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

scholarly journals Evaluating the effects of synthetic POM cycles and NAD kinase expression on fatty alcohol production in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Bonnie A McNeil ◽  
Charfeddine Khalifa ◽  
Anagha Krishnan ◽  
David T Stuart
Keyword(s):  
Fatty Alcohol ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Fatty Acid Biosynthesis ◽  
Nad Kinase ◽  
Acid Biosynthesis ◽  
Alcohol Production ◽  
Fatty Acid Biosynthesis Pathway ◽  
Kinase Expression ◽  
Background Expression

Abstract Background: NADPH-dependent enzymes play important roles in many anabolic reactions and the availability of redox cofactors can influence metabolic flux ultimately influencing titers of bioproducts produced by engineered microbial cells. This may be especially true of oleochemical production when carbon flux through the highly NADPH-dependent fatty acid biosynthesis pathway is increased. While pathway specific approaches are often applied to counter redox imbalance, a study evaluating generalized approaches to improved NADPH availability is lacking in Saccharomyces cerevisiae . Results: Here, we have created four unique synthetic Pyruvate-Oxaloacetate-Malate “POM” cycles consisting of either of the endogenous isoforms of pyruvate carboxylase ( PYC1 or PYC2 ), a modified version of malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and a truncated cytosolic form of the endogenous malic enzyme ( sMAE1 ). Only the POM cycle that combined expression of PYC1 , ‘MDH2 , and sMAE1 increased the titer of fatty alcohols produced; however, it did so in two unique fatty alcohol producing strains. In a FAS1 overexpression background, expression of this synthetic POM cycle increased fatty alcohol titers by 40% from 49.0 ± 2.2 mg/L to 68.6 ± 3.3 mg/L and showed similar results in a zwf1 deletion strain. The effect of overexpression of the endogenous NAD+ kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. We found that expression of POS5c resulted in an ~35% increase in fatty alcohol titer, while the overexpression of the UTR1 or YEF1 did not significantly influence titers. In these minimally engineered cells, combined overexpression of PYC1 , ‘ MDH2 , sMAE1 and POS5c did not further increase titers Conclusions: Overexpression of PYC1 in conjunction with ‘MDH2 and sMAE1 results in a synthetic POM cycle which can be utilized to improve fatty alcohol production in engineered strains of S. cerevisiae . Additionally, overexpression of a truncated version of POS5 ( POS5c ) results in similar increases in fatty alcohol production. These findings may serve to provide a generalized mechanism to increase NADPH production in engineered cells, resulting in increased bioproduct titers.

scholarly journals Revisiting Demand Rules for Gene Regulation

2015 ◽  
Author(s):  
Mahendra K. Prajapat ◽  
Kirti Jain ◽  
Debika Choudhury ◽  
Gauri S. Choudhary ◽  
Supreet Saini
Keyword(s):  
Gene Regulation ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Stochastic Simulations ◽  
Carbon Utilization ◽  
Acid Biosynthesis ◽  
Natural Distribution ◽  
Transcription Network ◽  
E Coli ◽  
Micro Level

Starting with Savageau's pioneering work from 1970s, here, we choose the simplest transcription network and ask: How does the cell choose a regulatory topology from the different available possibilities? We study the natural distribution of topologies at genome, systems, and micro-level in E. coli and perform stochastic simulations to help explain the differences in natural distributions. Analyzing regulation of amino acid biosynthesis and carbon utilization in E. coli and B. subtilis, we observe many deviations from the demand rules, and observe an alternate pattern emerging. Overall, our results indicate that choice of topology is drawn randomly from a pool of all networks which satisfy the kinetic requirements of the cell, as dictated by physiology. In short, simply, the cell picks "whatever works".

scholarly journals Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris

2004 ◽  
Vol 271 (12) ◽  
pp. 2462-2470 ◽  
Author(s):  
Aina Sola ◽  
Hannu Maaheimo ◽  
Katri Ylonen ◽  
Pau Ferrer ◽  
Thomas Szyperski
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Metabolic Flux ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis
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