1. Highly efficient and stereoselective biosynthesis of (2s,5s)-hexanediol with a dehydrogenase from saccharomyces cerevisiae.
Michael Katzberg, Werner Hummel, Marion Müller, Martin Bertau. Org Biomol Chem. 2010 Apr 7; 8(7): 1540-50. DOI: 10.1039/b920869k. PMID: 20237665.
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.
2. Laboratory evolution of pyrococcus furiosus alcohol dehydrogenase to improve the production of (2s,5s)-hexanediol at moderate temperatures.
Ronnie Machielsen, Annemarie Hendriks, Thomas Daussmann, John van der Oost, Stan J J Brouns, Hans-Georg Hennemann, Nicole G H Leferink. Extremophiles. 2008 Jul; 12(4): 587-94. DOI: 10.1007/s00792-008-0164-8. PMID: 18452026.
There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30 degrees C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30 degrees C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of approximately 1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30 degrees C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.
