Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

Abstract

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde ؉ NAD ؉ ؉ coenzyme A 7 acetyl coenzyme A ؉ NADH ؉ H ؉ ), was expressed in the gpd1⌬ gpd2⌬ strain, anaerobic growth was restored by supplementation with 2.0 g liter ؊1 acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD ϩ -dependent alcohol ...

Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

“…Whereas the use of E. coli mphF enabled acetate reduction in glucose fermentations in strains lacking GPD1 and GPD2 (ref. 35), mphF could not support the acetate reduction pathway in the presence of xylose (Fig. 2a).…”

“…A key glycerol production pathway has to be blocked to realize acetate reduction in the presence of glucose, as reported in a previous study where GPD1 and GPD2 were deleted and the E. coli mphF gene was overexpressed 35 . Further, although deletion of GPD1 and GPD2 improve ethanol yield by 13% in fermentation of 20 g l À 1 glucose and consumption of about 0.5 g l À 1 acetate, deletion of these two genes resulted in compromised cell growth and a substantial decrease in ethanol productivity 35 . The two enzymes Gpd1 and Gpd2 also have crucial roles in osmoregulation and phospholipid biosynthesis 36 .…”

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

“…4C), indicating improved flux through the xyloseassimilation pathway. Glycerol production, which represents a significant drain on ethanol production under anaerobic con- ditions (25,26), was not increased significantly (SI Appendix, Fig. S11).…”

Potential of aSaccharomyces cerevisiaerecombinant strain lacking ethanol and glycerol biosynthesis pathways in efficient anaerobic bioproduction

“…1 In addition, disrupting glycerol biosynthesis reactions enhanced ethanol production. 10 Therefore, glycerol biosynthesis reactions also contribute to maintaining the NADH/NAD + balance. In glycerol biosynthesis reactions, glycerol-3-phosphate dehydrogenases encoding GPD1 and GPD2 convert dihydroxyacetone phosphate to glycerol-3-phosphate and require NADH as a reducing power.…”