Adaptations in metabolism and protein translation give rise to the Crabtree effect in yeast

Aerobic fermentation, also referred to as the Crabtree effect in yeast, is a well-studied phenomenon that allows many eukaryal cells to attain higher growth rates at high glucose availability. Not all yeasts exhibit the Crabtree effect, and it is not known why Crabtree-negative yeasts can grow at rates comparable to Crabtree-positive yeasts. Here, we quantitatively compared two Crabtree-positive yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two Crabtree-negative yeasts, Kluyveromyces marxianus and Scheffersomyces stipitis, cultivated under glucose excess conditions. Combining physiological and proteome quantification with genome-scale metabolic modeling, we found that the two groups differ in energy metabolism and translation efficiency. In Crabtree-positive yeasts, the central carbon metabolism flux and proteome allocation favor a glucose utilization strategy minimizing proteome cost as proteins translation parameters, including ribosomal content and/or efficiency, are lower. Crabtree-negative yeasts, however, use a strategy of maximizing ATP yield, accompanied by higher protein translation parameters. Our analyses provide insight into the underlying reasons for the Crabtree effect, demonstrating a coupling to adaptations in both metabolism and protein translation.

CRABTREE POZİTİF VE CRABTREE NEGATİF MAYA TÜRLERİNDE GCR1 GENİNİN IN SILICO ANALİZİ

The use of fermentation in the presence of oxygen and at high glucose concentrations is referred to as the Crabtree effect. Yeast species that have the Crabtree effect are called Crabtree positive, and yeast species that do not have the Crabtree effect are called Crabtree negative. While Crabtree negative yeast strains are mostly used for heterologous protein production in the industrial field, Crabtree positive yeast strains are used to understand metabolic events in cancer cells. The genes encoding the enzymes involved in the glycolytic pathway in S. cerevisiae yeast cells are controlled by Gcr1p. Gcr1p binds to CT elements located in the promoter regions of glycolytic genes and activates their transcription. In our study, Crabtree positive and negative yeast strains containing Sc-Gcr1p similar proteins were determined, and protein similarity analyzes and promoter analyzes of genes encoding the relevant proteins in these yeast strains were compared in silico using different databases and analysis programs. For this purpose, SGD, UNIPROT, NCBI-Genome and Yeastract databases and BLASTp-NCBI, MEGA-X and Chromatin Folding V2 programs were used. Using the SGD database, 32 different yeast strains were identified that matched with Sc-Gcr1p. Five different Crabtree positive and 5 different Crabtree negative yeast strains were selected from these yeast strains and in silico analyzes were performed using these yeast strains. After protein analysis and promoter analysis, it was determined that the similarities and differences between yeast species were not specific for Crabtree positive and Crabtree negative yeast species, but varied between species.

Regulation of Eukaryote Metabolism: An Abstract Model Explaining the Warburg/Crabtree Effect

Adaptation of metabolism is a response of many eukaryotic cells to nutrient heterogeneity in the cell microenvironment. One of these adaptations is the shift from respiratory to fermentative metabolism, also called the Warburg/Crabtree effect. It is a response to a very high nutrient increase in the cell microenvironment, even in the presence of oxygen. Understanding whether this metabolic transition can result from basic regulation signals between components of the central carbon metabolism are the the core question of this work. We use an extension of the René Thomas modeling framework for representing the regulations between the main catabolic and anabolic pathways of eukaryotic cells, and formal methods for confronting models with known biological properties in different microenvironments. The formal model of the regulation of eukaryote metabolism defined and validated here reveals the conditions under which this metabolic phenotype switch occurs. It clearly proves that currently known regulating signals within the main components of central carbon metabolism can be sufficient to bring out the Warburg/Crabtree effect. Moreover, this model offers a general perspective of the regulation of the central carbon metabolism that can be used to study other biological questions.

Gene expression regulates metabolite homeostasis during the Crabtree effect: Implications for the adaptation and evolution of Metabolism

A key issue in both molecular and evolutionary biology has been to define the roles of genes and phenotypes in the adaptation of organisms to environmental changes. The dominant view has been that an organism’s metabolic adaptations are driven by gene expression and that gene mutations, independent of the starting phenotype, are responsible for the evolution of new metabolic phenotypes. We propose an alternate hypothesis, in which the phenotype and genotype together determine metabolic adaptation both in the lifetime of the organism and in the evolutionary selection of adaptive metabolic traits. We tested this hypothesis by flux-balance and metabolic-control analysis of the relative roles of the starting phenotype and gene expression in regulating the metabolic adaptations during the Crabtree effect in yeast, when they are switched from a low- to high-glucose environment. Critical for successful short-term adaptation was the ability of the glycogen/trehalose shunt to balance the glycolytic pathway. The role of later gene expression of new isoforms of glycolytic enzymes, rather than flux control, was to provide additional homeostatic mechanisms allowing an increase in the amount and efficiency of adenosine triphosphate and product formation while maintaining glycolytic balance. We further showed that homeostatic mechanisms, by allowing increased phenotypic plasticity, could have played an important role in guiding the evolution of the Crabtree effect. Although our findings are specific to Crabtree yeast, they are likely to be broadly found because of the well-recognized similarities in glucose metabolism across kingdoms and phyla from yeast to humans.

High Cell Density Cultivation of Saccharomyces cerevisiae with Intensive Multiple Sequential Batches Together with a Novel Technique of Fed-Batch at Cell Level (FBC)

High cell density cultivation (HCDC) is developed for the production of microbial biomasses and their products. They must be produced from high concentrations of substrate, e.g., glucose or sucrose. In batch culture, a high concentration of those sugars >40–50% (w/v) cannot efficiently be utilized because of a dissolved O2 limitation causing the Crabtree effect that produces toxic by-products, i.e., ethanol and/or acetate, that inhibit cell growth. To prevent this effect, the HCDC is conducted with the fed-batch strategies. However, it has many disadvantages, i.e., complicated operations. To overcome those problems, this study was designed to use a new, efficient C-source (carbon source) substrate, namely dextrin, an oligomer of glucose. It can be utilized by yeast at a very high concentration of ~100 g/L although using just batch cultivation. As it is gradually hydrolyzed to release glucose molecules and gradually assimilated into the cells as “fed-batch at the cell level” (FBC), it prevents the yeast cell system from undergoing the Crabtree effect. In this research, the types of medium, the types of sugar compared with dextrin, and the concentrations of yeast extract (YE) were studied. The batch production medium (BPM) with dextrin and YE performed very good results. The concentrations of dextrin for yeast cultivation were studied in the aerobic batch 5-L bioreactors. Its optimum concentration was at 90 g/L with 9 g/L of YE in 3× BPM. It was operated at 3 W/kg energy dissipation rate per unit mass (ε¯T) and 3 vvm airflow rate. Further, the intensive multiple sequential batch (IMSB) technique of high intensities of agitation speed and airflow was developed to achieve higher yield and productivity. The maximum values of cell biomass, specific growth rate, yield coefficient, productivity, and efficiency were at 55.17 g/L, 0.21 h−1, 0.54 g/g, 2.30 g/L/h, and 98.18%, respectively. The studies of cell growth kinetics, biochemical engineering mass balances, and fluid dynamics for the design of impeller speeds of the 5-L bioreactors during the cultivations of yeast using dextrin at the high concentrations were successful. The results can be used for the scale-up of bioreactor for the industrial production of yeast cell biomass at high concentrations.

Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica

The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of β-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L β-carotene. Both biomass and β-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP+/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, μm′, Yx/s′, and Yp/s′ was 0.527, 0.353, and 0.158, respectively. The μm′ was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Yx/s′ and Yp/s′ were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of β-carotene.

Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity of HepG2 cells to the purine derivative kinetin riboside

Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucose-induced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosis-inducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress.

A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer and neurological disorders. Therefore, unravelling the role of nutrients as signaling molecules and metabolites as well as their interconnectivity may provide a deeper understanding of how these conditions occur. Both signalling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism, we developed a hybrid model, combining Boolean model, describing the signalling layer and the enzyme constraint model accounting for metabolism using a regulatory network as a link. The model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression. We show that using this methodology one can investigat intrinsically different systems, such as signaling and metabolism, in the same model and gain insight into how the interplay between them can have non-trivial effects by showing a connection between Snf1 signaling and chronological lifespan by the regulation of NDE and NDI usage in respiring conditions. In addition, the model showed that during fermentation, enzyme utilization is the more important factor governing the protein allocation, while in low glucose conditions robustness and control is prioritized.Author summaryElucidating the complex relationship between nutrient-induced signaling and metabolism represents a key in understanding the onset of many different human diseases like obesity, type 3 diabetes, cancer and many neurological disorders. In this work we proposed a hybrid modeling approach, combining Boolean representation of singaling pathways, like Snf11, TORC1 and PKA with the enzyme constrained model of metabolism linking them via the regulatory network. This allowed us to improve individual model predictions and elucidate how single components in the dynamic signaling layer affect the steady-state metabolism. The model has been tested under respiration and fermentation, reveling novel connections and further reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression. Finally, we show a connection between Snf1 signaling and chronological lifespan by the regulation of NDE and NDI usage in respiring conditions.