Glycerol is of considerable importance in industrial yeast fermentations as the major by-product besides ethanol and carbon dioxide. In addition, glycerol accumulation is essential in osmotic stress resistance of yeast. With respect to wine fermentations higher glycerol yields are desired to improve product quality whereas lower glycerol production is expected to result in higher ethanol yields in the alcohol distillation industry. In baker's yeast production and application higher glycerol accumulation inside the cell is expected to improve the yeast's stress tolerance and performance during fermentation at high substrate osmolarity, during the drying process and during freesing and thawing of doughs.
Collaborative work between several of the partners in this proposal, involvlng active exchange of researchers, results and materials and evidenced by several common publications, has characterised major genetic components of glycero. biosynthesis and utilization as well as of glycerol uptake and efflux. Essential too s are now at hand to design novel yeast strains displaying either enhanced or reduced net glycerol production. For laboratory strains we have demonstrated that glycerol yields and internal glycerol accumulation can be altered greatly by changing the expression of only one or two genes. Hence, glycerol metabolism is a highly practicable model system for engineering metabolic fluxes even in genetically complex industrial yeasts. The first objective of this proposal is to engineer yeast strains towards higher glycerol production for use in the wine industry. This will be accomplished by first increasing the capacity of glycerol production and efflux. To further accelerate glycerol yields the glycolytic flux will be altered to provide higher substrate levels for glycerol production and/or glycerol re-utilization will be prevented. The metabolic, analytical and organoleptic consequences of enhanced glycero' production by the yeast will be studied. We will then design strategies to overcome possible adverse side effects in order to reach the best combination of yield and quality. The second objective is to engineer yeast strains such that they display reduced net glycerol synthesis for industrial alcohol production. Applicable approaches to be followed are aimed towards a reduced capacity to produce glycerol and/or to enhanced glycerol retention and re- utilization. The consequences of reduced glycerol formation on yield and purity of the ethanol produced will be investigated. If required, additional strategies will be developed to overcome unexpected side-effects.
The third objective is to engineer yeast strains displaying enhanced internal glycerol accumulation for use in the baker's yeast industry. Two complementary strategies will be followed: stimulation of glycerol production and improved retention of glycerol in the cell. Internal glycerol accumulation under conditions of industrial baker's yeast production and preservation of the glycerol content during downstream processing will be assessed. Our alternative strategy is to establish in Saccharomyces cerevisiae mechanisms used by non-conventional yeasts to achieve high osmotolerance by selection and expression of relevant genes from highly osmotolerant yeasts in S. cerevisiae. In order to obtain a complete understanding of the control of glycerol metabolism, especially with respect to industrial fermentation conditions, we will extend our collaborative work on the characterization of additional components involved in glycerol metabolism and transport. We will investigate in particular the essential role of glycerol metabolism under anaerobic conditions and its regulation by the cellular redoxstate. We will also further extend our studies on signal transduction pathways and transcriptional and post-transcriptional mechanisms in the control of glycerol production and efflux under osmotic stress.
To successfully execute the present proposal we have assembled a network comprising the world leaders in academic research on the biochemistry, physiology and molecular genetics of glycerol metabolism. We have combined this group with partners from industry-related research institutes and industrial laboratories directly involved with the wine industry, the alcohol distillery industry and industria baker's yeast production.
Funding SchemeCSC - Cost-sharing contracts
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