Means for reducing acetoin buildup in alcoholic fermentation media

9,017,989

12/451339

May 06, 2008

The invention relates to yeasts expressing the gene BDH1 coding for Bdh1p, characterized in that they catalyze, in an alcoholic fermentation medium, the reduction of acetoin into 2,3-butanediol by a rate at least twice higher than that of the initial stem. The invention can particularly be used for the fermentation of fruit juice and for producing 2,3-butanediol.

Dequin, Sylvie (Montpellier, FR); Ehsani, Maryam (Montpellier, FR); Fernández Gallegos, Maria Rosario (Barberà del Vallès, ES); Biosca, Josep A. (Barcelona, ES); Ortiz-Julien, Anne (Gagnac-sur-Garonne, FR)

1. An isolated Saccharomyces cerevisiae yeast strain in which the BDH1 gene encoding Bdh1p is overexpressed relative to the initial strain, wherein the overexpression results in an increase of the reduction of acetoin to 2,3-butanediol catalysed by Bdh1p at a rate that is at least twice that of the initial strain, said BDH1 gene is a mutated BDH1 gene comprising one or more mutations such that said gene encodes a Bdh1p protein in which one or more amino-acids are mutated in at least one of positions 221, 222 and 223 corresponding to a 221 SRS 223 fragment with reference to the Saccharomyces cerevisiae S288C strain, said mutation producing a Bdh1p protein having an increased affinity for NADPH as compared to the affinities of the Bdh1p protein without said mutation.

2. The yeast strain of claim 1 , wherein the protein encoded by the mutated BDH1 gene comprises 3 mutations at position 221, 222 and 223, corresponding to a 221 SRS 223 fragment with reference to the Saccharomyces cerevisiae S288C strain.

3. An isolated Saccharomyces cerevisiae yeast strain in which the BDH1 gene encoding Bdh1p is overexpressed relative to an initial strain, wherein the overexpression results in an increase of the reduction of acetoin to 2,3-butanediol catalyzed by Bdh1p at a rate that is at least twice that of the initial strain, wherein the BDH1 gene comprises mutations at positions 221, 222 and 223.

4. The yeast strain as claimed in claim 1 , characterized in that it overproduces glycerol.

5. The yeast strain as claimed in claim 4 , characterized in that it comprises an overexpressed GPD1 gene or an overexpressed GPD2 gene.

6. The yeast strain as claimed in claim 1 , characterized in that it is a genetically modified strain capable of reducing the amount of acetate produced in comparison with the strain as claimed in claim 5 .

7. The yeast strain as claimed in claim 6 , characterized in that the ALD6 gene has been deleted, and optionally copies thereof are also deleted.

8. The yeast strain as claimed in claim 3 , characterized in that it overproduces glycerol.

9. The yeast strain as claimed in claim 8 , characterized in that it comprises an overexpressed GPD1 gene or an overexpressed GPD2 gene.

4352/551

96/41888 December 1996

da Silva et al. Influence of plasmid origin and promoter strength in fermentations of recombinant yeast. Biotechnol Bioeng. Feb. 20, 1991;37(4):318-24. cited by examiner
International Search Report for PCT/IB2008/051761, mailed Oct. 6, 2008. cited by applicant
Gonzalez et al., “Characterization of a (2R, 3R)-2, 3-butanediol Dehydrogenase as the Saccharomyces cerevisiae Yal060W Gene Product. Disruption and Induction of the Gene”, Journal of Biological Chemistry, American Society of Biolochemical Biologists, Birmingham, US, vol. 275, No. 46, Nov. 2000, pp. 35876-35885. cited by applicant
Remize et al., “Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Stimulation of Fermentation and to a Stationary Phase”, Applied and Environmental Microbiology, vol. 65, No. 1, Jan. 1999, pp. 143-149. cited by applicant

edspgr.09017989