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.
Highlights
Acetic 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
Theoretical analysis of the stoichiometric impact of altering the cofactor specificity of 6‐PGDH Based on the assumption that the oxidative pentosephosphate pathway is the predominant source of NADPH in glucose-grown cultures of S. cerevisiae [4, 68], replacing the native NADP+-dependent 6-phosphogluconate dehydrogenase with an NAD+-dependent enzyme should result in an increased growth-coupled formation of cytosolic NADH
In the analysis, lumped stoichiometries for biosynthesis, NADPH formation via the pentose-phosphate pathway, NADH reoxidation through glycerol formation and redox-neutral, ATP-generating alcoholic fermentation were described by Eqs. 1–4, respectively [68]
Summary
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. 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. Industrial fermentation processes with S. cerevisiae are typically performed at pH values close to the pKa of acetic acid (4.75) This implies that a substantial fraction of the acid will be present in its non-dissociated form, which can diffuse across the yeast plasma membrane. At low to moderate concentrations of acetic acid (1–3 g L−1) and at pH values of 4–5, this increased demand for ATP results in lower biomass and glycerol yields and a higher ethanol yield on glucose in anaerobic cultures of S. cerevisiae [2, 27, 46]. In addition to the impact of acetic acid on intracellular pH homeostasis, intracellular accumulation of the acetate anion has been linked to increased oxidative stress and inhibition of key enzymes, such as aldolase [45], transaldolase and transketolase [23]
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