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Delft Researchers Create New Metabolic Pathway in Yeast to Boost Ethanol Yield from Biomass Waste

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


Ike Solem

This is clearly an area that will require a lot more focused effort in the whole-genome analysis area. It also shows a recurring theme - genetic engineering often disrupts some aspect of biochemical metabolism or basic gene function.

Solving those problems will require metabolic genetic engineering, which is the frontier in industrial microbiology these days - because while it is now easy to insert a gene that codes for a single enzyme, inserting a metabolic pathway if far more complicated. Evolution has probably already optimized the basic metabolic system of yeast, and tinkering with it is like messing around blindly under the hood of a car - the most likely result is a breakdown.

Sad to say, such research efforts in the U.S. are extremely limited - even the new DOE grants are not for the growth of public university research, but rather are directed to a small number of private-public partnerships outside of the academic system. The NSF and USDA likewise provide no funds for such research, leaving all biofuel researchers dependent on state grants or private support, which usually comes with many strings attached. This isn’t really a problem – unless new technologies are bought up and sat on instead of developed.

Maybe the climate is changing, however:

Green Tech America, Inc. is developing and commercializing a yeast-based cellulosic ethanol technology that was pioneered by Dr. Ho, Research Molecular Biologist/Group Leader of the Laboratory of Renewable Resources Engineering (LORRE) at Purdue University. (Earlier post.) During the 1980s and 1990s, researchers at LORRE altered the genetic structure of Saccharomyces yeast to enable the conversion of the two major sugars found in cellulosic materials—glucose and xylose—into ethanol.

The most notable thing about this is that while the technology was developed in the 1980s and 1990s, it has been sat on undeveloped for a good ten years at least, while Midwestern ethanol distillers have been allowed to switch to coal-powered ethanol distillation – thus giving the whole idea a bad name. Obviously, ethanol plants should be powered by wind and solar.



Don't forget that the wind stop blowing between 2000 and 2008.

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