Jorge L. Galazzo

Fermentation pathway kinetics and metabolic flux control in suspended and immobilized Saccharomyces cerevisiae

Abstract Measurements of rates of glucose uptake and of glycerol and ethanol formation combined with knowledge of the metabolic pathways involved in S. cerevisiae were employed to obtain in vivo rates of reaction catalysed by pathway enzymes for suspended and alginate-entrapped cells at pH 4.5 and 5.5. Intracellular concentrations of substrates and effectors for most key pathway enzymes were estimated from in vivo phosphorus-31 nuclear magnetic resonance measurements. These data show the validity in vivo of kinetic models previously proposed for phosphofructokinase and pyruvate kinase based on in vitro studies. Kinetic representations of hexokinase, glycogen synthetase, and glyceraldehyde 3-phosphate dehydrogenase, which incorporate major regulatory properties of these enzymes, are all consistent with the in vivo data. This detailed model of pathway kinetics and these data on intracellular metabolite concentrations allow evaluation of flux-control coefficients for all key enzymes involved in glucose catabolism under the four different cell environments examined. This analysis indicates that alginate entrapment increases the glucose uptake rate and shifts the step most influencing ethanol production from glucose uptake to phosphofructokinase. The rate of ATP utilization in these nongrowing cells strongly limits ethanol production at pH 5.5 but is relatively insignificant at pH 4.5.

Comparison of suspended and immobilized yeast metabolism using 31P nuclear magnetic resonance spectroscopy

31P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisiae anaerobic glucose metabolism in suspended and immobilized cells. Results show that cell entrapment in Ca-alginate beads alters cell metabolism compared to that in suspended cells. Assuming similar intracellular ionic strength, differences in intracellular phosphate chemical shift indicate that the internal pH of the immobilized cells is lower than the suspended cell internal pH. This result is consistent with higher ethanol production rates exhibited by immobilized yeast.

Redirection of cellular metabolism: analysis and synthesis

Achievement of optimal productivity and yields in bioprocesses using living cells generally requires redirection of cellular metabolic activity. Rarely is the native organism optimized with respect to process goals, thereby providing both the opportunity and the challenge of altering native metabolic function to achieve the most effective substrate utilization, cell growth, or product synthesis and release (or all of the above). The two main vehicles for metabolic manipulation-environmental control and alteration of the genetic constitution of the organism-are already evident in prior practice of bioprocessing art. However, until recently genetic manipulation was achieved primarily through random mutagenesis, and environmental manipulation was restricted to adjustment of solution composition during batch cultivation of cell suspensions. New developments in genetic technology and in engineering systems now provide the opportunity for more substantial and more carefully controlled and characterized manipulation of cellular DNA and environment. Using contemporary cloning techniques, the metabolic structure of a cell may be modified in a precise and wellcontrolled fashion by adding new proteins to the cell, by inhibiting or interfering with existing enzyme activities, by altering native control of expression of protein activities, and by amplifying particular protein activities already functional in the organism (e.g., see references 1-3). Furthermore, by the use of regulated replicators and expression

Application of linear prediction singular value decomposition for processingin vivo NMR data with low signal-to-noise ratio

Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by conventional methods requires samples with high cell density compared to that in growing cultures. Reasonably accurate intracellular concentration estimates from lower cell density samples can be obtained by treating the time-domain NMR data by linear prediction singular value decomposition (LPSVD) prior to Fourier transformation. Alternatively, application of LPSVD enables intracellular concentration estimates in less NMR acquisition time, improving time resolution in NMR measurements of intracellular transients.