Highly efficient and stereoselective biosynthesis of (2S,5S)-hexanediol with a dehydrogenase from Saccharomyces cerevisiae

Abstract

The enantiopure (2S,5S)-hexanediol serves as a versatile building block for the production of various fine chemicals and pharmaceuticals. For industrial and commercial scale, the diol is currently obtained through bakers' yeast-mediated reduction of 2,5-hexanedione. However, this process suffers from its insufficient space-time yield of about 4 g L(-1) d(-1) (2S,5S)-hexanediol. Thus, a new synthesis route is required that allows for higher volumetric productivity. For this reason, the enzyme which is responsible for 2,5-hexanedione reduction in bakers' yeast was identified after purification to homogeneity and subsequent MALDI-TOF mass spectroscopy analysis. As a result, the dehydrogenase Gre2p was shown to be responsible for the majority of the diketone reduction, by comparison to a Gre2p deletion strain lacking activity towards 2,5-hexanedione. Bioreduction using the recombinant enzyme afforded the (2S,5S)-hexanediol with >99% conversion yield and in >99.9% de and ee. Moreover, the diol was obtained with an unsurpassed high volumetric productivity of 70 g L(-1) d(-1) (2S,5S)-hexanediol. Michaelis-Menten kinetic studies have shown that Gre2p is capable of catalysing both the reduction of 2,5-hexanedione as well as the oxidation of (2S,5S)-hexanediol, but the catalytic efficiency of the reduction is three times higher. Furthermore, the enzyme's ability to reduce other keto-compounds, including further diketones, was studied, revealing that the application can be extended to alpha-diketones and aldehydes.

High-level production of (5S)-hydroxyhexane-2-one by two thermostable oxidoreductases in a whole-cell catalytic approach

“…Among those, Haberland et al [14] presented a continuous production process for enantiopure (2R,5R)-hexanediol with resting whole cells of Lactobacillus kefir reaching a space time yield (STY) of 64 g/L/d. For the production of the SS-diol, the best results were achieved with a purified dehydrogenase from Saccharomyces cerevisiae [17].…”

“…Since gluconic acid is produced during NADH regeneration, a high starting pH of 7.8 was chosen. To date, the highest published substrate concentrations for the production of 5-hydroxyhexane-2-one and 2,5-hexanediol by whole-cell or enzyme catalysis range from 50 to 110 mM [15,17,21,22]. Therefore, the conversion of 50 mM 2,5-hexanedione was investigated in the first biotransformation.…”

“…To our knowledge, no published data exist where an enzyme is resistant to similar high concentrations of 2,5-hexanedione. Therefore, the HBD is applicable in biotransformations with higher starting concentrations of 2,5-hexanedione than those used in other published biocatalytic processes where in maximum 110 mM 2,5-hexanedione were initially applied [15][16][17]21,22].…”

“…The reduction to (5S)-hydroxyhexane-2-one was performed by Katzberg et al [22] with whole-cells of S. cerevisiae L13 in a batch mode. However, like in all other published batch reactions [15,17,21], only rather low substrate concentrations of 20-110 mM 2,5-hexanedione were applied, resulting in only low final product concentrations. To obtain high final concentrations of (5S)-hydroxyhexane-2-one in batch processes, the application of a S-selective dehydrogenase bearing a high activity and a high resistance towards 2,5-hexanedione is desirable.…”

“…Various studies have already shown the applicability of enzymes or whole-cell catalysts in the reduction of 2,5-hexanedione to the corresponding enantiopure diols [14][15][16][17][18] which are essential building blocks for chiral catalysts [19]. Among those, Haberland et al [14] presented a continuous production process for enantiopure (2R,5R)-hexanediol with resting whole cells of Lactobacillus kefir reaching a space time yield (STY) of 64 g/L/d.…”