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Exposed to changes in their environment, microorganisms will adapt their phenotype, including metabolism, to ensure survival. To understand the adaptation principles, resource allocation-based approaches were successfully applied to predict an optimal proteome allocation under (quasi) steady-state conditions. Nevertheless, for a general, dynamic environment, enzyme kinetics will have to be taken into account which was not included in the linear resource allocation models. To this end, a resource-dependent kinetic model was developed and applied to the model organism Saccharomyces cerevisiae by combining published kinetic models and calibrating the model parameters to published proteomics and fluxomics datasets. Using this approach, we were able to predict specific proteomes at different dilution rates under chemostat conditions. Interestingly, the approach suggests that the occurrence of aerobic fermentation (Crabtree effect) in S. cerevisiae is not caused by space limitation in the total proteome but rather an effect of constraints on the mitochondria. When exposing the approach to repetitive, dynamic substrate conditions, the proteome space was allocated differently. Less space was predicted to be available for non-essential enzymes (reserve space). This could indicate that the perceived “overcapacity” present in experimentally measured proteomes may very likely serve a purpose in increasing the robustness of a cell to dynamic conditions, especially an increase of proteome space for the growth reaction as well as of the trehalose cycle that was shown to be essential in providing robustness upon stronger substrate perturbations. The model predictions of proteome adaptation to dynamic conditions were additionally evaluated against respective experimentally measured proteomes, which highlighted the model’s ability to accurately predict major proteome adaptation trends. This proof of principle for the approach can be extended to production organisms and applied for, Applied Sciences, BT/Industrial Microbiology

Exposed to changes in their environment, microorganisms will adapt their phenotype, including metabolism, to ensure survival. To understand the adaptation principles, resource allocation-based approaches were successfully applied to predict an optimal proteome allocation under (quasi) steady-state conditions. Nevertheless, for a general, dynamic environment, enzyme kinetics will have to be taken into account which was not included in the linear resource allocation models. To this end, a resource-dependent kinetic model was developed and applied to the model organism Saccharomyces cerevisiae by combining published kinetic models and calibrating the model parameters to published proteomics and fluxomics datasets. Using this approach, we were able to predict specific proteomes at different dilution rates under chemostat conditions. Interestingly, the approach suggests that the occurrence of aerobic fermentation (Crabtree effect) in S. cerevisiae is not caused by space limitation in the total proteome but rather an effect of constraints on the mitochondria. When exposing the approach to repetitive, dynamic substrate conditions, the proteome space was allocated differently. Less space was predicted to be available for non-essential enzymes (reserve space). This could indicate that the perceived “overcapacity” present in experimentally measured proteomes may very likely serve a purpose in increasing the robustness of a cell to dynamic conditions, especially an increase of proteome space for the growth reaction as well as of the trehalose cycle that was shown to be essential in providing robustness upon stronger substrate perturbations. The model predictions of proteome adaptation to dynamic conditions were additionally evaluated against respective experimentally measured proteomes, which highlighted the model’s ability to accurately predict major proteome adaptation trends. This proof of principle for the approach can be extended to production organisms and applied for, BT/Industriele Microbiologie

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.

Biofilm formation is a survival strategy for microorganisms facing a hostile environment. Under biofilm, bacteria are better protected against antibacterial drugs and the immune response, increasing treatment difficulty, as persistent populations recalcitrant to chemotherapy are promoted. Deciphering mechanisms leading to biofilms could, thus, be beneficial to obtain new antibacterial drug candidates. Here, we show that mycobacterial biofilm formation is linked to excess glycerol adaptation and the concomitant establishment of the Crabtree effect. This effect is characterized by respiratory reprogramming, ATP downregulation, and secretion of various metabolites including pyruvate, acetate, succinate, and glutamate. Interestingly, the Crabtree effect was abnormal in a mycobacterial strain deficient for Cpn60.1 (GroEL1). Indeed, this mutant strain had a compromised ability to downregulate ATP and secreted more pyruvate, acetate, succinate, and glutamate in the culture medium. Importantly, the mutant strain had higher intracellular pyruvate and produced more toxic methylglyoxal, suggesting a glycolytic stress leading to growth stasis and consequently biofilm failure. This study demonstrates, for the first time, the link between mycobacterial biofilm formation and the Crabtree effect., SCOPUS: ar.j, info:eu-repo/semantics/published

When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the genetic network that determines the duration of the lag phase has not been studied in much detail. Here, we performed a genome-wide Bar-Seq screen to identify genetic determinants of the Saccharomyces cerevisiae glucose-to-galactose lag phase. The results show that genes involved in respiration, and specifically those encoding complexes III and IV of the electron transport chain, are needed for efficient growth resumption after the lag phase. Anaerobic growth experiments confirmed the importance of respiratory energy conversion in determining the lag phase duration. Moreover, overexpression of the central regulator of respiration, HAP4, leads to significantly shorter lag phases. Together, these results suggest that the glucose-induced repression of respiration, known as the Crabtree effect, is a major determinant of microbial fitness in fluctuating carbon environments.IMPORTANCE The lag phase is arguably one of the prime characteristics of microbial growth. Longer lag phases result in lower competitive fitness in variable environments, and the duration of the lag phase is also important in many industrial processes where long lag phases lead to sluggish, less efficient fermentations. Despite the immense importance of the lag phase, surprisingly little is known about the exact molecular processes that determine its duration. Our study uses the molecular toolbox of S. cerevisiae combined with detailed growth experiments to reveal how the transition from fermentative to respirative metabolism is a key bottleneck for cells to overcome the lag phase. Together, our findings not only yield insight into the key molecular processes and, BT/Industrial Microbiology

When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the genetic network that determines the duration of the lag phase has not been studied in much detail. Here, we performed a genome-wide Bar-Seq screen to identify genetic determinants of the Saccharomyces cerevisiae glucose-to-galactose lag phase. The results show that genes involved in respiration, and specifically those encoding complexes III and IV of the electron transport chain, are needed for efficient growth resumption after the lag phase. Anaerobic growth experiments confirmed the importance of respiratory energy conversion in determining the lag phase duration. Moreover, overexpression of the central regulator of respiration, HAP4, leads to significantly shorter lag phases. Together, these results suggest that the glucose-induced repression of respiration, known as the Crabtree effect, is a major determinant of microbial fitness in fluctuating carbon environments.IMPORTANCE The lag phase is arguably one of the prime characteristics of microbial growth. Longer lag phases result in lower competitive fitness in variable environments, and the duration of the lag phase is also important in many industrial processes where long lag phases lead to sluggish, less efficient fermentations. Despite the immense importance of the lag phase, surprisingly little is known about the exact molecular processes that determine its duration. Our study uses the molecular toolbox of S. cerevisiae combined with detailed growth experiments to reveal how the transition from fermentative to respirative metabolism is a key bottleneck for cells to overcome the lag phase. Together, our findings not only yield insight into the key molecular processes and, BT/Industriele Microbiologie

Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism’s natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine-tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.

[Background] Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. [Results] Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. [Conclusions] REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines.

Constraint-based modeling is largely used in computational studies of metabolism. We propose here a novel approach that aims to identify ensembles of flux distributions that comply with one or more target phenotype(s). The methodology has been tested on a small-scale model of yeast energy metabolism. The target phenotypes describe the differential pattern of ethanol production and O2 consumption observed in "Crabtree-positive" and "Crabtree-negative" yeasts in changing environment (i.e., when the upper limit of glucose uptake is varied). The ensembles were obtained either by selection among sampled flux distributions or by means of a search heuristic (genetic algorithm). The former approach provided indication about the probability to observe a given phenotype, but the resulting ensembles could not be unambiguously partitioned into "Crabtree-positive" and "Crabtree-negative" clusters. On the contrary well-separated clusters were obtained with the latter method. The cluster analysis further allowed identification of distinct groups within each target phenotype. The method may thus prove useful in characterizing the design principles underlying metabolic plasticity arising from evolving physio-pathological or developmental constraints. © 2014 Springer Science+Business Media.

Under aerobic conditions, most yeasts such as Kluyveromyces lactis, prefer the respiratory pathway and some, such as Saccharomyces cerevisiae prefer less energy efficient fermentative pathway for their energy metabolism. These two metabolic strategies are also known as Crabtree negative and Crabtree positive respectively, and the evolution of the latter has lately been explained by the “make-accumulate-consume” life strategy. Scientists have for more than a century tried to elucidate the mechanism behind the physiology and the evolution of the peculiar respiro-fermentation trait. During the last decades, comparative genomics approaches have enabled the reconstruction of the evolutionary history of yeast, and several evolutionary events have been identified and postulated to have contributed to the development of the respiro-fermentative lifestyle in the Saccharomyces lineage. However, many of these inspiring studies have been verified with reference species only. Therefore, as parts of my thesis I conducted large-scale physiology studies of more than 40 yeast species and their central carbon metabolism under controlled conditions, in bioreactors. This was done in order to map the evolution of aerobic fermentation in yeast belonging to the Saccharomyces lineage that span over 200 million years of yeast evolution. This evolutionary blueprint, which most likely will be an invaluable information source of primary data for future in silico studies on the evolution of Crabtree effect, has already verified the importance of evolutionary events, such as promoter rewiring, chromatin relaxation, whole genome duplication, gene duplication and lateral gene-transfers. I further propose a mechanism that provides an explanation for the origin of the respiro-fermentative lifestyle in yeast, and how this was subsequently, through a multistep process developed into the Crabtree effect as we know it in the modern yeasts today, such as S. cerevisiae and its sister species.

A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate. Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate. If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases. These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells

Diabetic pregnancy is associated with an increased incidence of congenital malformations. The teratologicalmechanism may be related to an alteration of embryonic organogenesis by maternal metabolic derangementsduring early gestation. Mitochondria, the site of biological oxidation in the embryo, may be directly affectedby the disturbed maternal metabolism. The embryonic mitochondria may respond to an increased metabolicload by producing an increased amount of free oxygen radicals, which, in diabetic pregnancy, has beensuggested to disturb embryonic morphogenesis. The present study demonstrated that embryonic mitochondriaexposed to maternal diabetes in vivo or to high concentrations of glucose, pyruvate, β-hydroxybutyrate andα-ketoisocaproate in vitro during organogenesis showed a pronounced morphological alteration, a high-amplitude swelling. The swelling of mitochondria was eliminated both in vivo by maternal ingestion of theantioxidants, vitamin E or BHT, and in vitro by inhibition of mitochondrial uptake of excess pyruvate withCHC, thus suggesting that the swelling of embryonic mitochondria was a result of oxidative damage causedby free oxygen radicals produced by the mitochondria themselves. When embryonic neural tissue wasincubated with high concentration of glucose, oxygen uptake of the tissue was inhibited and glucoseutilization was stimulated, indicating a Crabtree effect. The changed metabolic state induced by highconcentration of glucose was associated with a generation of superoxide. Embryos of diabetic rats showed alow oxygen uptake and a low glucose utilization. Superoxide production was undetectable in these embryos,indicating an impaired mitochondrial function. High concentration of β-hydroxybutyrate induced differentmetabolic alterations in embryonic neural tissue during early and late organogenesis. Superoxide generationmight be induced by the ketone body during early organogenesis. Thus, the teratogenic mechanism of theketone body may differ, depend

Applied Sciences

Applied Sciences

Applied Sciences

Applied Sciences

Applied Sciences