Glucose uptake and ethanol and glycerol production are about twice as fast in immobilized cells as in suspended cells. The observed differences in intracellular components between suspended and immobilized cells are qualitatively similar to the differences observed for cells grown in suspension. In alginate-grown cells, glucose uptake limits ethanol production in both suspended and immobilized cells.
There is a 5% decrease in the flux control coefficient of glucose uptake in immobilized cells due to an increase in the rate of glucose uptake. An analysis of the predicted effects of genetic manipulation to improve the rate of ethanol production in the yeast S.
Introduction
Metabolic manipulation of organisms through environmental control is one of the most attractive features of immobilized cell systems. a) Cell immobilization uncouples cell growth and product formation. However, an understanding of the interactions between the cell and the immobilization process is essential. Immobilization can change the metabolic activity of the cells because physical stresses that densely packed cells exert on each other and on the support are not present in traditional fermentations.
This trial-and-error approach has usually been carried out using ill-defined systems, making extrapolation of the results difficult. The experimental information is used in Chapter 4, combined with knowledge of the metabolic pathways involved in S.
In vivo nuclear magnetic resonance
In the immobilized cell experiments, a perfusion device allowed the medium to circulate through the NMR tube containing the cells immobilized with Ca-alginate. Therefore, the rate of glucose uptake is also greater for immobilized cells than for suspended cells. Estimates of intermediate near-steady-state concentrations obtained in this way for suspended and immobilized cells are shown in Table II.
There are substantial differences in glucose-6-phosphate, fructose-6-phosphate and 3-phosphoglycerate levels in suspended and immobilized cells. However, the qualitative differences between suspended and immobilized cells remain mostly the same as observed at pH.
Study of glucose metabolic kinetics of
Cell growth in the liquid phase is evident 6 hours after the start of the experiment. The growth rate of immobilized cells is slower than that of suspended cells (µ= 0.41 h-1), which is in agreement with several other studies. (6-8). This indicates that the intracellular pH of the immobilized cell is initially lower, probably due to the collapse of the transmembrane potential during the immobilization process.
The intracellular pH of immobilized cells (6.8) is slightly lower than the intracellular pH of suspended cells (6.9) as indicated by the peak position of Pfn. Also, the qualitative differences in the peak shape of SP indicate a different composition of SP between suspended and immobilized cells. Units are in mmol per L of cell volume, assuming that 1.6 g of wet cells contains 1.0 mL of cells. (14) Sugar-phosphate concentrations were obtained using an in vivo deconvolution method. (13) The AT Pa + ADPA score was integrated to estimate ATP+ ADP levels.
Immobilization of cells can increase the permeability of the cell membrane, making it more permeable to protons. If the biocatalyst conditions (suspended or immobilized cells) are compared in Table III, the rate of ethanol production is twice as high in the immobilized cells, independent of the cell growth conditions. Comparing growth conditions (in suspension or in trap), ethanol is produced 1.5 times faster in cells grown in alginate than in cells grown in suspension, independent of the biocatalyst configuration.
Total sugar phosphate levels are higher in suspension-cultured cells than in alginate-cultured cells (12 mM and 5 mM, respectively), but the composition of the sugar phosphates is similar in both systems. An understanding of cell-environment interactions is essential when designing and applying immobilized cell biocatalysts. Cooper T., "Transport in Saccharomyces cerevisiae" In: The molecular biology of yeast, metabolism and gene expression", p.
Fermentation pathway kinetics and
Measurements of the rates of glucose uptake and glycerol and ethanol formation combined with knowledge of the metabolic pathways involved in S. Calculation of control coefficients requires a detailed knowledge of the intracellular state as well as the in vivo values of system parameters such as pH intracellular and allosteric effector levels. The metabolic pathway from glucose to ethanol, glycerol, and polysaccharides-... is shown in Figure 1. Enzymes of intermediate steps not shown are assumed to catalyze very fast reactions so that equilibria are maintained at all times.
Thus, measuring the rates of glucose uptake (V 1n ), glycerol production (Va01 ), and ethanol production (V EtOH ) provides most of the internal reaction rates under steady-state conditions. Estimation of the in vivo parameters for the reactions in the model pathway is done using the results of experiments of cells grown in suspension (Table I). Processes that change the total amount of adenine nucleotides are considered negligible over the time frame of the experiments and are not included in the model.
AN is a free model parameter, but the same value is used for both suspended and immobilized cells in all experimental conditions shown in Table I because the cells are in the same initial state. P; it is not included in the kinetic expression because its concentration is almost the same in all experiments of Table I (about 10 mM). Therefore, knowing the intracellular pH, an estimate of the allosteric constant can be made for in vivo conditions.
Product inhibition by N ADH is also included, as well as the inhibitory effects of AMP, ADP and ATP.(39,39) The maximum velocity (VGA.PD) of this enzyme is assumed to be the same for both suspended and immobilized cells. Pyruvate kinase activity is also affected by pH through changes in the maximum velocity (through Lo) and in the binding affinity (KR,PEP) of phosphoenolpyruvate.(4o,4i) The functionality of PK on pH was obtained from in vitro data (41l with using the kinetic expression from Table VIL Figure 6 shows Lo and KR,PEP as a function of pH Similar model to phosphofructokinase (Table V) with fructose-1,6-diphosphate as allosteric activator.(34,35) Parameter values from references (35) and (40) The pH dependencies of Lo and KR,PEP were determined from in vitro data<41.
In summary, the only adjustable parameters of the model are the intracellular glucose concentration (Gin), the ratio between reduced and oxidized NAD (N ADH/N AD+) and the overall rate constant of ATP consumption (kATPase)·. Experimental ethanol production rate was used to obtain Vj;';[ and the kinetic expression in Table VII and in vivo NMR pH and metabolite concentration data were used to Vj]:0• Point ( •) was used to calculate VJJ'f [" which was assumed to be the same for all other experiments.
Analysis of the potential for improving
This model is used in this chapter to study which enzymes (steps) in the pathway need to be changed to improve ethanol production. This chapter presents an analysis of how flow control coefficients and ethanol production would change if the activity of any of the steps mentioned above were to change. An increase in the rate of glucose uptake (V~n) would decrease the control coefficient (crn) of this rate, which would improve ethanol production.
It is reasonable to predict from the flux control coefficients of Table I that the improvement in ethanol production rate will be most pronounced for cells suspended at extracellular pH 4.5, because crn has a value close to unity under those conditions, thus indicating that glucose uptake is almost completely limiting flux throughout the pathway. As expected from Table I, the improvement in ethanol production that changes the rate of glucose uptake is more significant for suspended cells than for immobilized cells. From these results it can be predicted that an increase in VPFK from the in vivo value would not have a significant effect on ethanol production in suspended cells, while the effect would be more pronounced in immobilized cells.
This is more clearly observed in Figure 4, where the ethanol production rate is plotted as a function of maximum PFC activity. This is because ATPase does not contribute to the flux control of the pathway at this extracellular pH (Table I). Therefore, there is no effect on the ethanol production rate by increasing kAT Pa. On the other hand, ethanol production increases with pH. % in suspended cells and 7% in immobilized cells by increasing kATPa•• by 30% over the in vivo values reported in Chapter 4.
For example, the relative increase in ethanol production due to increasing VJn is greater in suspended cells than in immobilized cells because glucose uptake is the main control point in suspended cells, whereas several steps share the flux control in immobilized cells. There are four steps that share the control of ethanol production in immobilized cells at pH-glucose uptake, phosphofructokinase, polysaccharide storage and ATP consumption. Calculation of flux control coefficients in the alginate-grown cells indicates that glucose uptake limits the ethanol production rate in both suspended and immobilized cells.
Conclusions
Data on the yeast glucose carrier indicate that interconversion between the two forms of the carrier is the most likely rate-limiting step in transport. To properly apply the LPSVD method to Bruker spectrometer quadratic FID data, which sample the real and imaginary channels sequentially, (18). Most discrepancies using the FFT are around 20%, even for the 7 min scan (Figure 2-a).
LPSVD provides estimates of metabolite concentrations within 10% of the reference values using acquisition times of 5 minutes. The agreement between the two methods is in this case slightly better than in the low cell density experiments because the signal-to-noise ratio of the reference signal is higher. Comparison of the downfield area of a 15-sec. scan of a 40% (v / v) yeast suspension performing steady state glucose catabolism.
This research was sponsored by the Energy Conversion and Utilization Technology (ECUT) Program of the USA. At the desired time, the metabolism of the cell suspension was stopped by freezing the cells with liquid nitrogen and the intracellular components were extracted by adding perchloric acid. . 31P NMR spectra of the cellular suspensions were obtained in the Fourier transform mode at 202.46 MHz on a Bruker AM-500 NMR spectrometer at 20°.
The supernatant was transferred to another pre-chilled test tube and neutralized with cold 2 M KHCO 3 • pH was checked with litmus paper. Estimates of the levels of the sugar phosphates were determined by a systematic procedure based on 31 P NMR measurements. In vitro sugar phosphate levels were determined by integration of the peaks in the sugar phosphate region of spectra of cell extracts.
