ADH from Rhodococcus ruber expressed in Arxula adeninivorans for the synthesis of 1-(S)-phenylethanol

• Synthesis of 1-(S)-phenylethanol by RrADH produced in Arxula adeninivorans.
• Coexpression of BmGDH and RrADH in A. adeninivorans.
• Isopropanol and BmGDH coupled regeneration.

The RrADH gene of Rhodococcus ruber coding for (S)-specific alcohol dehydrogenase (RrADH) was overexpressed in the yeast Arxula adeninivorans and used for the synthesis of enantiomerically pure alcohols.The substrates acetophenone, 2,5-hexandione and 2-nonanon used for this synthesis, were reduced by RrADH to produce S-configured alcohols. Regeneration of the cofactor, NADH, was required for the reaction and this was provided by using isopropanol as a second ADH substrate to reduce NAD+ or by cloning the glucose dehydrogenase gene from Bacillus megaterium (BmGDH) into the yeast which regenerated NADH by oxidizing glucose. Expressing both RrADH and BmGDH in the yeast provided a strain that could synthesize 1-(S)-phenylethanol from acetophenone with NADH being regenerated by the oxidation of glucose.Both bioreduction systems led to the synthesis of pure (S) enantiomer of 1-phenylethanol, but only the enzyme coupled approach reduced 40 mM acetophenone completely in 150 min. 75 mg of 98% pure product could be isolated from 20 ml.In conclusion the synthesis potential of the RrADH expressed in A. adeninivorans is very promising for 1-(S)-phenylethanol synthesis.

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