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Applied and Environmental Microbiology, May 2009, p. 3196-3205, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02157-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing, Low-Alcohol Saccharomyces cerevisiae{triangledown}

Maryam Ehsani,1,3 Maria R. Fernández,2 Josep A. Biosca,2 Anne Julien,3 and Sylvie Dequin1*

INRA, UMR1083, F-34060 Montpellier, France,1 Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain,2 Lallemand SAS, 19 Rue Briquetiers, F-31700 Blagnac, France3

Received 17 September 2008/ Accepted 19 March 2009

Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3°.

* Corresponding author. Mailing address: INRA, UMR 1083, 2 Place Viala, 34060 Montpellier Cedex 1, France. Phone: (33) 4 99 61 25 28. Fax: (33) 4 99 61 28 57. E-mail: dequin{at}supagro.inra.fr

{triangledown} Published ahead of print on 27 March 2009.

Applied and Environmental Microbiology, May 2009, p. 3196-3205, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02157-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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