Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway

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There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine's sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.

Section: Discussionmentioning

confidence: 91%

mentioning

confidence: 99%

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

Tilloy

Ortiz‐Julien

Dequin

2014

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“…However, Cadiere et al (5) found that yeast strains exhibiting higher carbon fluxes through this pathway showed no effect on ethanol production. Mutants lacking PGI1 and therefore channeling carbon through the pentose phosphate pathway were not able to sustain growth even when overexpressing ZWF1, indicating that another reaction or factor limits flux through this pathway (24).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Gene Modification Strategies for the Development of Low-Alcohol-Wine Yeasts

Varela

Kutyna

Solomon

et al. 2012

ABSTRACTSaccharomyces cerevisiaehas evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO2. Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene,GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.

“…The simplest method is to use growth rate as a selection pressure by performing serial subcultures of a target strain under specific culture conditions (27,28). This approach was applied for isolating S. cerevisiae mutants that show improved resistance against stress conditions, such as oxidative, freeze-thawing, high-temperature, and ethanol stresses (29).…”

Section: Discussionmentioning

confidence: 99%

Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Kim

Lin

et al. 2013

f Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular ␤-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K m and 4-fold higher V max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains. Ethanol production from cellulosic biomass not only is sustainable but also does not cause ethical issues like corn-or sugarbased ethanols do (1). The economic production of ethanol from cellulosic biomass requires substantial improvements in all unit operations, such as physical pretreatment, chemical or enzymatic depolymerization, and fermentation. The efficient hydrolysis of cellulose and the utilization of mixed sugars (glucose and xylose) present in cellulosic hydrolysates are especially necessary for producing cellulosic ethanol on a commercial scale (2-4).Fungal cellulases, which are widely used for degrading cellulose, exhibit weak ␤-glucosidase activity. The low ␤-glucosidase activity can lead to an accumulation of cellobiose because of its slow degradation of cellobiose into glucose during simultaneous saccharification and fermentation (SSF). The accumulation of cellobiose during SSF reduces the overall rate and efficiency of cellulose hydrolysis because cellobiose is an inhibitor of cellulases. Therefore, supplementation with bacterial or fungal ␤-glucosidase is necessary to enhance cellulose hydrolysis in spite of the disadvantages in the cost of doing so. In order to reduce the enzyme costs from supplementation with ␤-glucosidase, engineered Saccharomyce...