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Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae. In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.

Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae