TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 1085-1093 ◽  
Author(s):  
Lars M. Blank ◽  
Uwe Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Glucose Uptake ◽  
Environmental Conditions ◽  
Specific Growth ◽  
Tca Cycle ◽  
Glucose Sensing ◽  
The Tca Cycle ◽  
Uptake Rates ◽  
Wide Range

Metabolic responses of Saccharomyces cerevisiae to different physical and chemical environmental conditions were investigated in glucose batch culture by GC-MS-detected mass isotopomer distributions in proteinogenic amino acids from 13C-labelling experiments. For this purpose, GC-MS-based metabolic flux ratio analysis was extended from bacteria to the compartmentalized metabolism of S. cerevisiae. Generally, S. cerevisiae was shown to have low catabolic fluxes through the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. Notably, respiratory TCA cycle fluxes exhibited a strong correlation with the maximum specific growth rate that was attained under different environmental conditions, including a wide range of pH, osmolarity, decoupler and salt concentrations, but not temperature. At pH values of 4·0 to 6·0 with near-maximum growth rates, the TCA cycle operated as a bifurcated pathway to fulfil exclusively biosynthetic functions. Increasing or decreasing the pH beyond this physiologically optimal range, however, reduced growth and glucose uptake rates but increased the ‘cyclic’ respiratory mode of TCA cycle operation for catabolism. Thus, the results indicate that glucose repression of the TCA cycle is regulated by the rates of growth or glucose uptake, or signals derived from these. While sensing of extracellular glucose concentrations has a general influence on the in vivo TCA cycle activity, the growth-rate-dependent increase in respiratory TCA cycle activity was independent of glucose sensing.

scholarly journals Correlation between TCA cycle flux and glucose uptake rate during respiro-fermentative growth of Saccharomyces cerevisiae

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3827-3837 ◽  
Author(s):  
Jan Heyland ◽  
Jianan Fu ◽  
Lars M. Blank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Uptake ◽  
Production Rate ◽  
Environmental Conditions ◽  
Glucose Repression ◽  
Single Gene ◽  
Tca Cycle ◽  
Acetate Production ◽  
The Tca Cycle ◽  
Uptake Rates

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using 13C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO2 allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g−1 h−1), while the highest acetate production rate of 2.1 mmol g−1 h−1 was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l−1 , pH 5.0, 30 °C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g−1 h−1 to about 1.7 mmol g−1 h−1 was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO2 to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.

scholarly journals Proteome allocations change linearly with specific growth rate of Saccharomyces cerevisiae under glucose-limitation

2021 ◽  
Author(s):  
Jianye Xia ◽  
Benjamin Sánchez ◽  
Yu Chen ◽  
Kate Campbell ◽  
Sergo Kasvandik ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Specific Growth Rate ◽  
Growth Rates ◽  
Specific Growth ◽  
Cell Factory ◽  
Functional Protein ◽  
Wide Range ◽  
The Absolute ◽  
Specific Growth Rates

Abstract Saccharomyces cerevisiae is widely used as a cell factory and it is therefore important to understand how it organizes key functional parts when cultured under different conditions. Here we performed a multi-omics analysis of S. cerevisiae by culturing the strain under a wide range of specific growth rates using glucose as the sole limited nutrient. At these different conditions we measured the absolute transcriptome, the absolute proteome, the phosphproteome, and the metabolome. Most functional protein groups showed linear dependence on the cell specific growth rate. Proteins engaged with translation showed a perfect linear increase with the specific growth rate, while glycolysis and chaperone proteins showed a linear decrease at respiratory conditions. Glycolytic enzymes and chaperones, however, show decreased phosphorylation with increasing specific growth rates, resulting in an overall increased activity that is associated with increased flux through these pathways. Further analysis showed that proteome allocation was primarily determined at the transcriptome level. Finally, using enzyme constraint genome scale modeling we found that enzyme usage play an important role for controlling flux in amino acid biosynthesis.

scholarly journals Influence of organic acids and organochlorinated insecticides on metabolism of Saccharomyces cerevisiae

2005 ◽  
pp. 207-215 ◽  
Author(s):  
Dusanka Pejin ◽  
Vesna Vasic
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Sugar Beet ◽  
Specific Growth Rate ◽  
Glycogen Content ◽  
Specific Growth ◽  
Raw Materials ◽  
Stress Factors ◽  
Adaptive Mechanism ◽  
Ribonucleic Acids

Saccharomyces cerevisiae is exposed to different stress factors during the production: osmotic, temperature, oxidative. The response to these stresses is the adaptive mechanism of cells. The raw materials Saccharomyces cerevisiae is produced from, contain metabolism products of present microorganisms and protective agents used during the growth of sugar beet for example the influence of acetic and butyric acid and organochlorinated insecticides, lindan and heptachlor, on the metabolism of Saccharomyces cerevisiae was investigated and presented in this work. The mentioned compounds affect negatively the specific growth rate, yield, content of proteins, phosphorus, total ribonucleic acids. These compounds influence the increase of trechalose and glycogen content in the Saccharomyces cerevisiae cells.

scholarly journals Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Thomas Perli ◽  
Dewi P.I. Moonen ◽  
Marcel van den Broek ◽  
Jack T. Pronk ◽  
Jean-Marc Daran
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Nicotinic Acid ◽  
Specific Growth ◽  
Pantothenic Acid ◽  
B Vitamins ◽  
Fast Growth ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
B Vitamin

AbstractQuantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

scholarly journals Anaplerotic reactions active during growth of Saccharomyces cerevisiae on glycerol

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Joeline Xiberras ◽  
Mathias Klein ◽  
Celina Prosch ◽  
Zahabiya Malubhoy ◽  
Elke Nevoigt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Specific Growth Rate ◽  
Pyruvate Carboxylase ◽  
Reference Strain ◽  
Specific Growth ◽  
Glyoxylate Cycle ◽  
Maximum Specific Growth Rate ◽  
The Glyoxylate Cycle ◽  
Anaplerotic Reactions

ABSTRACT Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h−1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.

scholarly journals Inclusion of maintenance energy improves the intracellular flux predictions of CHO

2020 ◽  
Author(s):  
Diana Széliová ◽  
Jerneja Štor ◽  
Isabella Thiel ◽  
Marcus Weinguny ◽  
Michael Hanscho ◽  
...  
Keyword(s):  
Growth Rates ◽  
High Throughput Screening ◽  
Energy Demand ◽  
Metabolic Flux ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Metabolic Model ◽  
Maintenance Energy ◽  
Uptake Rates ◽  
Wide Range

AbstractChinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by pFBA using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.

scholarly journals KINETIC EVALUATION OF ETHANOL-TOLERANT THERMOPHILE Geobacillus thermoglucosidasius M10EXG FOR ETHANOL PRODUCTION

2016 ◽  
Vol 10 (1) ◽  
pp. 24
Author(s):  
Eny Ida Riyanti ◽  
Peter L. Rogers
Keyword(s):  
Growth Rate ◽  
Specific Growth Rate ◽  
Ethanol Production ◽  
Yeast Extract ◽  
Biomass Yield ◽  
Specific Growth ◽  
Kinetic Evaluation ◽  
Geobacillus Thermoglucosidasius ◽  
Low Level ◽  
Wide Range

Thermophiles are challenging to be studied for ethanol production using agricultural waste containing lignocellulosic materials rich in hexose and pentose. These bacteria have many advantages such as utilizing a wide range of substrates, including pentose (C5) and hexose (C6). In ethanol production, it is important to use ethanol tolerant strain capable in converting lignocellulosic hydrolysate. This study was aimed to investigate the growth profile of ethanol-tolerant thermophile Geobacillus thermoglucosidasius M10EXG using a defined growth medium consisted of single carbon glucose (TGTV), xylose (TXTV), and a mixture of glucose and xylose (TGXTV), together with the effect of yeast extract addition<br />to the media. The experiments were conducted at the School of Biotechnology and Biomolecular Sciences of The University of New South Wales, Australia on a shake flask fermentation at 60°C in duplicate experiment. Cultures were sampled every two hours and analised for their kinetic parameters including the maximum specific growth rate (µmax), biomass yield (Yx/s), ethanol and by-product yields (acetate and L-lactate) (Yp/s), and the doubling time (Td). Results showed that this strain was capable of growing on minimal medium containing glucose or xylose as a single carbon source. This strain utilized glucose and xylose simultaneously (co-fermentation), although there was glucose repression of xylose at relatively low glucose concentration (0.5% w/v), particularly when yeast extract (0.2% w/v) was added to the medium. The highest biomass yield was obtained at 0.5 g l-1 on glucose medium; the yield increased when yeast extract was added (at 0.59 g l-1). The highest specific growth rate of 0.25 was obtained in the phase I growth when the strain was grown on a mixture of glucose and xylose (0.5% : 0.5% w/v) medium. Diauxic growth was shown on the mixture of glucose, xylose, and yeast extract. The strain produced low level of ethanol (0.1 g l-1), as well as low level (0.2 g l-1) of by-products (L-lactate and acetate) after 15 hours. The results suggests its potential application for fermenting lignocellulosic agricultural wastes for ethanol production.

scholarly journals Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Jasmine M. Bracher ◽  
Erik de Hulster ◽  
Charlotte C. Koster ◽  
Marcel van den Broek ◽  
Jean-Marc G. Daran ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Specific Growth Rate ◽  
Specific Growth ◽  
Baker’S Yeast ◽  
Industrial Biotechnology ◽  
Baker's Yeast ◽  
Laboratory Evolution ◽  
Content Type ◽  
Study Laboratory

ABSTRACT Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h−1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h−1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h−1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h−1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

Siderophore production by Fusarium venenatum A3/5

2002 ◽  
Vol 30 (4) ◽  
pp. 696-698 ◽  
Author(s):  
M. G. Wiebe
Keyword(s):  
Growth Rate ◽  
Specific Growth Rate ◽  
Environmental Conditions ◽  
Continuous Flow ◽  
Specific Growth ◽  
Chemostat Culture ◽  
Siderophore Production ◽  
Batch Cultures ◽  
Chemostat Cultures ◽  
Fusarium Venenatum

Fusarium venenatum A3/5 was grown in iron-restricted batch cultures and iron-limited chemostat cultures to determine how environmental conditions affected siderophore production. The specific growth rate in iron-restricted batch cultures was 0.22 h−1, which was reduced to 0.12 h−1 when no iron was added to the culture. Derit in iron-limited chemostat culture was 0.1 h−1. Siderophore production was correlated with specific growth rate, with the highest siderophore production occurring at D = 0.08 h−1 and the lowest at D = 0.03 h−1. Siderophore production was greatest at pH 4.7 and was significantly reduced at pHs above 6.0. Siderophore production could be enhanced by providing insoluble iron instead of soluble iron in continuous flow cultures.

Sign in / Sign up

Export Citation Format

Share Document