Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

In vivo reconstitution of autophagy in Saccharomyces cerevisiae

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.     

In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope

We present a robust second-generation BioScope: a system for continuous perturbation experiments. Firstly, the BioScope design parameters (i.e., pressure drop, overall oxygen (O2) and carbon dioxide (CO2) mass transfer, mean residence time distribution and plug flow characteristics) were evaluated. The average overall mass transfer coefficients were estimated to be 1.8E-5 m s(-1) for O2 and 0.34E-5 m s(-1) for CO2. It was determined that the O2/CO2 permeable membrane accounted for 75% and 95% of the overall resistance for O2 and CO2, respectively. The Peclet number (Pe) of the system was found to be >500 for liquid flow rates between 1 and 4 ml min(-1), ensuring plug flow characteristics. Secondly, steady-state intracellular metabolite concentrations obtained using direct rapid sampling from the fermentor were compared with those obtained by rapid sampling via the pre-perturbation sample port of the BioScope. With both methods the same metabolite levels were obtained. Thirdly, glucose perturbation experiments were carried out directly in the fermentor as well as in the BioScope, whereby steady-state Saccharomyces cerevisiae cells from a glucose/ethanol limited chemostat were perturbed by increasing the extracellular glucose concentration from 0.11 to 2.8 mM. Intracellular and extracellular metabolite levels were measured within a time window of 180 s. It was observed that the dynamic metabolite concentration profiles obtained from both perturbations were nearly the same, with the exception of the C4 metabolites of the TCA cycle, which might be due to differences in culture age.     

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.     

In vivo investigations of glucose transport in Saccharomyces cerevisiae

In the present study, the glucose transport into the yeast Saccharomyces cerevisiae has been investigated. The approach suggested is based on a rapid sampling technique for studying the dynamic response of the yeast to rapid changes in extracellular glucose concentrations. For this purpose a concentrated glucose solution has been injected into a continuous culture at steady state growth conditions resulting in a shift of the extracellular glucose level. Samples have been taken every 5 s for determination of extracellular glucose and intracellular glucose-6-phosphate concentrations. Attempts to fit the experimental observations with simulations from existing models failed. The mechanism then proposed is based on a facilitated diffusion of glucose superimposed by an inhibition of glucose-6-phosphate. The use of the so-called in vivo approach suggested in this article appears to be proper, because the investigations can be performed at defined physiological states of the microbial cultures. Furthermore, the experimental observations are not being corrupted by the preparation of the samples for the transport studies as it happens during radioactive measurements. (c) 1996 John Wiley & Sons, Inc.     

In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels

The dynamics of galactose metabolism in Saccharomyces cerevisiae was studied by analyzing the metabolic response of the CEN.PK 113-7D wild-type strain when exposed to a galactose pulse during aerobic growth in a galactose-limited steady-state cultivation at a dilution rate of 0.097 h(-1). A fast sampling technique and subsequent methanol-chloroform/solid phase extractions were applied for in vivo measurements of the dynamic changes of the AMP, ADP, ATP levels and the sugar phosphates of the Leloir pathway. The ATP level was found to be significantly lower for yeast growing under galactose limitation (0.37 +/- 0.05 micromol/g CDW) than what has been reported for growth under glucose limitation. The galactose pulse of 5.58 mM was consumed within 40 min (t = 40) and 7 min after the pulse was added cell growth stopped. Subsequently, the cells started to grow and at t = 30 the specific growth rate had recovered to half the steady-state growth rate (0.047 h(-1)). To evaluate the change in flux distribution at steady state and during the galactose transient, a stoichiometric model describing the aerobic metabolism of S. cerevisiae was set up for quantification of the metabolic fluxes. At t = 7 the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal-1P was measured, which may be responsible for a toxic metabolic response in S. cerevisiae. The increase in the Gal-1P concentration is intensified by the low affinity of Gal7 towards Gal-1P and, hence, under the physiological conditions examined Gal7 seems to exert control over flux through the Leloir pathway.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.     

Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80. The organism had a mu max of 0.31 h-1 and a Ks for glucose of 0.55 mM. At a dilution rate of 0.10 h-1, a maximal yield of 0.10 g biomass (g glucose)-1 was observed. The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 0.10 h-1, the yield of the S. cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition. The major difference between the two yeast strains was that S. cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid. The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase. The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed. This resulted in a reduction in yield and an increase in specific ethanol production. Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth. Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes.     

Circadian rhythmicity during prolonged chemostat cultivation of Neurospora crassa

Following exposure to light and attainment of steady-state in the chemostat, Neurospora was grown in constant conditions of darkness at 25 degrees C for 6 days. Biomass samples were taken every 4h for the extraction of RNA and protein, and the state of the circadian clock was assessed by assaying the levels of three rhythmically expressed mRNAs; frequency (frq), antisense frq (qrf) and clock-controlled gene-14 (ccg-14), and by monitoring the clock-controlled rhythm of sporulation. Our results indicate that the Neurospora clock continued to run in the chemostat. This is the longest reported time that Neurospora has been grown in a chemostat in filamentous form and opens up the possibility of studying the response of Neurospora to a range of stimuli in the absence of confounding effects due to; alterations in growth rate, aging, and changing conditions of the growth medium.     

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast–famine conditions to characterize the long-term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long-term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast–famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.     

In vivo nuclear magnetic resonance analysis of immobilization effects on glucose metabolism of yeast Saccharomyces cerevisiae

Fermentation rates and intracellular compositions have been determined for alginate-entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose-1,6 diphosphate, glucose-6-phosphate, and 3-phosphoglycerate and in internal pH. Carbon-13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.     

In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae

The in vivo dynamics of the pentose phosphate pathway has been studied with transient experiments in continuous culture of Saccharomyces cerevisiae. Rapid sampling was performed with a special sampling device after disturbing the steady state with a pulse of glucose. The time span of observation was 120 s after the pulse. During this short time period the dynamic effect of protein biosynthesis can be neglected. The metabolites of interest (glucose 6-phosphate, NADP, NADPH, 6-phosphogluconate, and MgATP2-) we determined with enzymatic assays and HPLC. The experimental observations were then used for the identification of kinetic rate equations and parameters under in vivo conditions. In accordance with results from in vitro studies the in vivo diagnosis supports an ordered Bi-Bi mechanism with noncompetitive inhibition by MgATP2- for the enzyme glucose-6-phosphate dehydrogenase. In the case of 6-phosphogluconate dehydrogenase an ordered Bi-Ter mechanism with a competitive inhibition by MgATP2- has been found. Because the MgATP2- concentration decreases abruptly after the pulse of glucose the inhibitory effect vanishes and the flux through the pentose phosphate pathway increases. This regulation phenomenon guarantees the balance of fluxes through glycolysis and pentose phosphate pathway during the dynamic time period.     

Dynamics of anaerobic growth of Saccharomyces cerevisiae in the chemostat.

A kinetic Monod model has been used to describe the dynamic response of a continuous stirred tank fermentor (CSTF) to changes in dilution rate. A general analytical solution of a linearized model was obtained. Experimental results (Vairo et al. 1977) of continuous anaerobic culture of Saccharomyces cerevisiae have verified the model quantitatively. For step disturbances on the dilution rate the responses of biomass concentration and the outlet substrate concentration were calculated on a digital computer and compared with the experimental data.     

Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae.

We have investigated whether central nitrogen metabolism may influence the triggering of ethanol fermentation in Saccharomyces cerevisiae strain CEN.PK122 grown in the presence of different N-sources (ammonia, glutamate, or glutamine) under conditions in which the carbon to nitrogen (C : N) ratio was varied. An exhaustive quantitative evaluation of yeast physiology and metabolic behavior through metabolic flux analysis (MFA) was undertaken. It is shown that ethanol fermentation is triggered at dilution rates, D (growth rate), significantly lower (D=0.070 and 0.074 h(-1) for glutamate and glutamine, respectively, and D=0.109 h(-1) for ammonia) under N- than C-limitation (approximately 0.18 h(-1) for all N-sources). A characteristic specific rate of glucose influx, q(Glc), for each N-source at Dc, i.e., just before the onset of respirofermentative metabolism, was determined (approximately 2.0, 1.5, and 2.5, for ammonia, glutamate, and glutamine, respectively). This q(Glc) was independent of the nutritional limitation though dependent on the nature of the N-source. The onset of fermentation occurs when this "threshold q(Glc)" is overcome. The saturation of respiratory activity appears not to be associated with the onset of fermentation since q(O(2)) continued to increase after Dc. It was remarkable that under respirofermentative conditions in C-limited chemostat cultures, the glucose consumed was almost completely fermented with biomass being synthesized from glutamate through gluconeogenesis. The results obtained show that the enzyme activities involved in central nitrogen metabolism do not appear to participate in the control of the overflow in carbon catabolism, which is driven toward ethanol production. The role of nitrogen metabolism in the onset of ethanol fermentation would rather be realized through its involvement in setting the anabolic fluxes directed to nitrogenous macromolecules. It seems that nitrogen-related anabolic fluxes would determine when the threshold glucose consumption rate is achieved after which ethanol fermentation is triggered.