Author:
Aalst Aafke C.A. van,Geraats Ellen H.,Jansen Mickel L.A.,Mans Robert,Pronk Jack T.
Abstract
AbstractIn anaerobicSaccharomyces cerevisiaecultures, NADH-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as ‘redox-sink’ and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation. An alternative glycerol-reduction strategy bypasses the oxidative reaction in glycolysis by introducing phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (RuBisCO). For optimal performance in industrial settings, yeast strains should ideally first fully convert acetate and, subsequently, continue low-glycerol fermentation via the PRK-RuBisCO pathway. However, anaerobic batch cultures of a strain carrying both pathways showed inferior acetate reduction relative to a strain expressing only the A-ALD pathway. Complete A-ALD-mediated acetate reduction by a dual-pathway strain, grown anaerobically on 50 g L-1glucose and 5 mmol L-1acetate, was achieved upon reducing PRK abundance by a C-terminal extension of its amino-acid sequence. Yields of glycerol and ethanol on glucose were 55% lower and 6% higher, respectively, than those of a non-engineered reference strain. The negative impact of the PRK-RuBisCO pathway on acetate reduction was attributed to sensitivity of the reversible A-ALD reaction to intracellular acetaldehyde concentrations.
Publisher
Cold Spring Harbor Laboratory
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