Physiological and fitness phenotypes of yeast overexpressing glycolytic enzymes

Evaluating the adaptive significance of genetic enzyme polymorphisms remains a controversial issue in evolutionary biology. One model, based on metabolic control theory, assumes that if fitness and pathway flux covary and are both maximized a hyperbolic function relates fitness to single-step activity changes. The model predicts that large activity increases will negligibly affect fitness/flux compared to large decreases. Consequently, most allozymes occupy the plateau of this function and are selectively neutral. Evidence supporting this model has been obtained using the wild-type fully-induced state as the activity maximum. I report an experimental system that circumvents this limitation and tests the model more rigorously.;DNA fragments complementing lesions in glycolysis were overexpressed in Saccharomyces cerevisiae on high-copy plasmids. These included: hexokinaseB, phosphoglucose-isomerase, phosphofructokinase and pyruvate kinase. Activities ranged from four- to ten-fold over either wild-type or wild-type transformed with vector only. Biochemical, physiological, and fitness parameters were measured in exponential and stationary-phase batch cultures. These included: levels of certain glycolytic intermediates, glucose conversion to ethanol and glycogen, yield biomass in dry mass and protein, maximum growth rate, stationary-phase density, heat-shock sensitivity and sporulation efficiency.;Intermediate pools during exponential phase, flux to ethanol, yield biomass, and heat-shock sensitivity do not vary significantly among treatments and controls. However, each treatment does present at least one distinct phenotype. HexokinaseB transformants overaccumulate trehalose in stationary phase. Phosphoglucose-isomerase transformants hyperaccumulate glycogen during mid-exponential phase. Phosphofructokinase transformants fail to accumulate glycogen during stationary phase. Pyruvate kinase transformants demonstrate lower growth rate, higher stationary-phase density, sporulate poorly, and fail to accumulate glycogen during stationary phase. Like phosphofructokinase transformants, they present hexose phosphate profiles that suggest overexpression impedes gluconeogenesis by creating a futile cycle. Transformants carrying Tn5 disruptions of plasmid-borne PYK or plasmid-borne PGI present control phenotypes.;Metabolic networks appear intrinsically buffered to large, constituitive, single-step increases in the activity of "housekeeping" enzymes. Nevertheless, such increases have measurable physiological impact. For at least one enzyme, pyruvate kinase, this translates into potential fitness phenotypes. It follows that higher-order character states of allozyme variants that show comparable differences may not always be predictable using models based on metabolic control theory.