Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae

Abstract

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.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

“…The slower consumption of xylose as compared with glucose might also play a role in increasing isobutanol production. Recently, it has been shown that implementing a flux valve increases the yield of isobutanol from engineered S. cerevisiae (Tan, Manchester, & Prather, ). Endogenous hexokinases GLK1 and HXK2 were deleted alongside downregulation of HXK1 using a doxycycline‐inducible repressor, leading to a reduced rate of glucose phosphorylation and lowered glycolytic flux.…”

“…Metabolites were extracted and quantified from cells grown in the mid-exponential phase as described in the materials and methods. Each column represents one biological replicate while each row represents a single metabolite [Color figure can be viewed at wileyonlinelibrary.com] Recently, it has been shown that implementing a flux valve increases the yield of isobutanol from engineered S. cerevisiae (Tan, Manchester, & Prather, 2016). Endogenous hexokinases GLK1 and HXK2 were deleted alongside downregulation of HXK1 using a doxycycline-inducible repressor, leading to a reduced rate of glucose phosphorylation and lowered glycolytic flux.…”

Into new territory: improved microbial synthesis through engineering of the essential metabolic network

“…One set of tools for dynamic metabolic engineering are genetic "toggle" switches, which enable the inducible repression of genes including essential enzymes responsible for competitive metabolic fluxes [33][34][35][36][37][38][39][40]. A common essential and competitive metabolic pathway is the citric acid or tricarboxylic acid (TCA) cycle.…”

Engineering Biomolecular Switches for Dynamic Metabolic Control

“…Moreover, the specific growth rate of engineered E. coli can be reduced by up to 50% without altering final biomass accumulation. The same strategy was then employed to develop a metabolite valve in S. cerevisiae for control of glycolytic flux through the central carbon metabolism [72]. This was demonstrated by diverting glucose flux away from glycolysis into a model pathway, gluconate, in a hexokinase 2 and glucokinase 1 deleted strain.…”