Résumé
Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium chain dehydrogenases/reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p, in different forms: purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p) to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the "delitto perfetto" methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells), yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p towards the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.