The relationship of glycolytic/gluconeogenic intermediates in brewing yeast (Saccharomyces uvarum) fermentations to growth (1984)

The objective of this study has been to understand the metabolic interrelationship between yeast growth, regulation of glycolytic/gluconeogenic flux and accumulation of glycosyl donors for polysaccharide synthesis in brewing yeast (Saccharomyces uvarum) fermentations.Loss of fermenting power of a brewing yeast population may be created by a condition that inhibits growth by limiting amino acid formation and protein synthesis.In commercial strains of S. uvarum this loss may be transitory, or, if not corrected, may ulti~ately lead to yeast degeneration.The potential industrial impact is realised for fermentation systems which may limit yeast growth, eg.continuous systems, use of pressure and, particularly, systems utilizing immobilised cells.In order to chronicle the associated metabolic events occurring under conditions which promote cellular proliferation and under conditions which terminate growth in the exponential growth phase, a specific analytical strategy was adopted.It was considered at the outset of this work that the multivalent nature of the glycolytic/gluconeogenic controlling allosteric enzymes, often complicated by numerous intracellular modulators, prevented reproducing in vitro, a relatable in vivo enzymatic situation.To gain an accurate overall picture of intracellular glycolytic/gluconeogenic control, it was, therefore, decided to measure the levels of pertinent cellular intermediates and effectors.To this end, the development of isotachophoresis and bioluminescence as measurement techniques provided the specificity and sensitivity (picomole range) required to examine a wide range of intracellular nucleotides, organic acids and hexose and triose phosphates.Experiments indicated that growth-limiting conditions are associated with high ATP, GTP, citrate and phosphoenolpyruvate levels and low levels of AMP, fructose-1,6-bisphosphate and pyruvate in agreement with the proposed regulatory mechanisms of phosphofructokinase I (EC 2.7.1.11),pyruvate kinase (EC 2.7.1.40),phosphoenolpyruvate carboxykinase (EC 4.1.1.49),fructose-1,6-bisphosphatase (EC 3.1.3.11) and aldolase (EC 4.1.2.13).The aggregate effect of these changes is in favour of inhibiting phosphofructokinase I and pyruvate kinase activity and activating phqsphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase, thus orientating the glycolytic pathway in a gluconeogenic direction.Regulation at the phosphofructokinase I/fructose-1.,6-bisphosphatasejunction created a build-up of fructose-6-phosphate and subsequently glucose-6-phosphate which, by feed-back, cooperatively inhibited glucose utilization.Measurements showed that the end-point of gluconeogenesis in yeast and/or inhibition of phosphofructokinase I is not glucose-6-phosphate in yeast as has been previously reported.Under growth-limiting conditions, the glycogenic pathway was utilized as far as the glycogen substrate, uridine-5'diphosphoglucose (UDPG), creating an augmented UDPG pool.Concomitantly, a build-up of the cell wall precursor, uridine-5'-diphosphate-N-acetylglucosarnine, occurred.This indicated that control of glutamine fructose-6-phosphate transaminase (EC 5.3.1.19)by UDP acetylglucosamine, was incompletely suppressed, at least as far as the yeast strains utilized were concerned.Under non-proliferating conditions, the augmented UDPG pool was not channelled to glycogen.Moreover, under these conditions the level of glycogen decreased to a base level or to a level where fonnation and consumption are virtually equal.Thus, glycogen synthesis and degradation in brewing strains of s. uvarum appears to be a highly regulated, physiological process, dependent not only on the availability of UDPG and glycogen as respective substrates.It is proposed that allosteric and covalent regulation of the interconvertible phosphorylated and dephosphorylated fonns of glycogen synthetase (EC 2.4.1.11)and phosphorylase (EC 2.4.1.1)is of major significance.In this regard, the intracellular levels of the adenine nucleotides and glucose-6-phosphate would appear to play a particularly important role.A regulation model is proposed of glycogen synthesis and degradation when exponential growth is inhibited in S. uvarum.The physiological meaning of the increased UDPG concentration under the described conditions is unclear.It may be that the maintenance of a UDPG pool is a more efficient mechanism for the yeast cell to tenuously support viability and integrity even though, under the test procedures, the cell is ultimately unable to ferment.