Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6

Abstract

BackgroundAcetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption.ResultsReplacement of the native NADP+-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD+-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15 % increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP+-dependent acetaldehyde dehydrogenase, led to a 39 % increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44 % increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13 % increase in the ethanol yield on glucose.ConclusionsThe combination of NAD+-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0465-z) contains supplementary material, which is available to authorized users.

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

“…Strain IMX992 ( GPD1 GPD2 sga1::eutE ) showed an acetate consumption of 3.35 mmol (g biomass) −1 , which was only 0.92 mmol (g biomass) −1 higher than the acetate consumption by the GPD1 GPD2 reference strain. Conversely, under identical conditions, strain IMX888 ( gpd1Δ gpd2::eutE ) showed an acetate consumption of 6.92 mmol (g biomass) −1 in a previous study [29]. Consistent with its marginally higher acetate consumption, glycerol production by strain IMX992 decreased only slightly, from 9.19 to 8.28 mmol glycerol (g biomass) −1 , relative to strain IME324 (Table 3).…”

“…Increased adaptation phases, reflecting a non-genetic population heterogeneity, are well documented in acetate-stressed S. cerevisiae cultures [68, 69]. Recent studies on engineered A-ALD expressing S. cerevisiae strains in low- and medium-osmolarity cultures [26, 29] lend further support to the conclusion that deletion of ALD6 is a key step in engineering efficient pathways for acetate reduction in S. cerevisiae .…”

Influence of Datura stramonium Leaf Extract on Antioxidants and Activities of Metabolic Enzymes of Trigonella foenum-graecum and Lepidium

“…6PGDH (EC.1.1.1.44) was determined according to the method of Papapetridis et al, (2016). The method is dependent on the conversion of NAD + /NADP + to NADH/NADPH and measuring absorbance at 340 nm.…”

A robust flow cytometry-based biomass monitoring tool enables rapid at-line characterization of S. cerevisiae physiology during continuous bioprocessing of spent sulfite liquor

“…HMF and furfural have a still not fully explored inhibitory effect on yeast growth and ethanol productivity [9]. While acetic acid can be co-utilized as an additional carbon source in addition to sugars by commonly used biotechnological production hosts Saccharomyces cerevisiae and Escherichia coli [10][11][12], it has a strong influence on the cytosolic pH and can negatively influence viability [2,13]. Beside those challenges, the biorefinery of spent sulfite liquor provides the opportunity to produce sustainable biofuels by valorization of the waste stream.…”