Researchers from Delft University of Technology have engineered the yeast Saccharomyces cerevisiae to increase ethanol yield from biomass waste by eliminating production of glycerol (glycerol production is essential to reoxidize NADH produced in biosynthetic processes), reoxidizing NADH instead by the reduction of acetic acid to ethanol. A paper on their work was published online 13 November in the journal Applied and Environmental Microbiology.
Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass—a pre-treatment for fermentation—and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates, the authors note. This new metabolic engineering strategy is thus a triple win, says principal researcher Jack Pronk: “no glycerol formation, higher ethanol yields and consumption of toxic acetate”.
Researchers have estimated that up to 4% of the sugar feedstock in typical industrial ethanol processes is converted into glycerol. Several other metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae with lower or no success.
The goal of the present study is to investigate whether the engineering of a linear pathway for the NADH dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provides a stoichiometric basis for elimination of glycerol production during industrial ethanol production.
The S. cerevisiae genome already contains genes encoding acetyl-Coenzyme A synthetase and NAD+-dependent alcohol dehydrogenases. To complete the linear pathway for acetic acid reduction, we expressed an NAD+-dependent, acetylating acetaldehyde dehydrogenase from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae.—Medina et al.
The researchers found that they were indeed able to reduce the glycerol yield to zero, while the apparent ethanol yield on glucose has increased to 62 C-mol%, representing a theoretical 18% increase relative to the ethanol yield of the reference strain grown on glucose as the sole carbon source.
While their work provides a proof of principle that, stochiometrically, the role of glycerol as a redox sink for anaerobic growth of S. cerevisiae can be fully replaced by a linear pathway for NADH-dependent reduction of acetate to ethanol, the authors note that several issues remain to be addressed before industrial implementation would be possible:
Growth and product formation in the engineered strain were significantly slower than in the reference strain, due to a number of possible factors.
Glycerol, which protects yeast cells at high extracellular osmolarity, is likely to be relevant in industrial fermentations with high initial sugar concentrations. Analysis needs to be done to analyze osmotic stress in anaerobic cultures unable to produce glycerol. “Such research should, ultimately, address the question whether robust industrial yeast strains can be constructed that do not produce glycerol.”
The Delft yeast researchers, who applied for a patent on their invention, hope to intensively collaborate with industrial partners to accelerate its industrial implementation.
Guadalupe Medina et al. (2009) Elimination of glycerol production in anaerobic cultures of Saccharomyces cerevisiae engineered for use of acetic acid as electron acceptor. Applied and Environmental Microbiology doi: 10.1128/AEM.01772-09 10.1128/AEM.01772-09