Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase.

Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and 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 toward 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.

Biocatalytic production of alpha-hydroxy ketones and vicinal diols by yeast and human aldo-keto reductases.

The α-hydroxy ketones are used as building blocks for compounds of pharmaceutical interest (such as antidepressants, HIV-protease inhibitors and antitumorals). They can be obtained by the action of enzymes or whole cells on selected substrates, such as diketones. We have studied the enantiospecificities of several fungal (AKR3C1, AKR5F and AKR5G) and human (AKR1B1 and AKR1B10) aldo-keto reductases in the production of α-hydroxy ketones and diols from vicinal diketones. The reactions have been carried out with pure enzymes and with an NADPH-regenerating system consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase. To ascertain the regio and stereoselectivity of the reduction reactions catalyzed by the AKRs, we have separated and characterized the reaction products by means of a gas chromatograph equipped with a chiral column and coupled to a mass spectrometer as a detector. According to the regioselectivity and stereoselectivity, the AKRs studied can be divided in two groups: one of them showed preference for the reduction of the proximal keto group, resulting in the S-enantiomer of the corresponding α-hydroxy ketones. The other group favored the reduction of the distal keto group and yielded the corresponding R-enantiomer. Three of the AKRs used (AKR1B1, AKR1B10 and AKR3C1) could produce 2,3-butanediol from acetoin. We have explored the structure/function relationships in the reactivity between several yeast and human AKRs and various diketones and acetoin. In addition, we have demonstrated the utility of these AKRs in the synthesis of selected α-hydroxy ketones and diols.

Role of Saccharomyces cerevisiae oxidoreductases Bdh1p and Ara1p in the metabolism of acetoin and 2,3-butanediol.

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.