Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae

Abstract

BackgroundGlycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance.Results GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway.ConclusionsDeletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0791-3) contains supplementary material, which is available to authorized users.

Characterization of Saccharomyces bayanus CN1 for Fermenting Partially Dehydrated Grapes Grown in Cool Climate Winemaking Regions

“…The link between these two metabolites in S. cerevisiae under hyperosmotic stress is based on a redox balance of the NAD + /NADH system. The formation of glycerol generates NAD + [15,17,43]. Acetic acid production from acetaldehyde reduces NAD + to NADH through the activity of a NAD + -dependent aldehyde dehydrogenase, and corrects the redox shift [17,18,44].…”

MOMO - multi-objective metabolic mixed integer optimization: application to yeast strain engineering

“…The proposed deletions of genes involved in glycerol metabolism as a means of improve ethanol production is of particular interest, since several studies have demonstrated that indeed modulation of the activity of this pathway can contribute, at different extents, to improve ethanol production in yeast (reviewed in [29][30][31]). It can be reasoned that the reduced activity of pathways consuming pyruvate and acetaldeyde can contribute to decrease the diversion of pyruvate, creating a surplus of these metabolites that can then be channeled for ethanol synthesis.…”

Momo – Multi-Objective Metabolic mixed integer Optimization: application to yeast strain engineering

“…The proposed deletions of genes involved in glycerol metabolism as a means of improve ethanol production is of particular interest, since several studies have demonstrated that indeed modulation of the activity of this pathway can contribute, at different extents, to improve ethanol production in yeast (reviewed in [26][27][28]). It can be reasoned that the reduced activity of pathways consuming pyruvate and acetaldeyde can contribute to decrease the diversion of pyruvate, creating a surplus of these metabolites that can then be channeled for ethanol synthesis.…”