Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol

The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l⁻¹ADH and 370 U l⁻¹G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l⁻¹ADH and 170 U l⁻¹GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH–LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP⁺, makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l⁻¹ day⁻¹or 406 g kg⁻¹dry cell weight (dcw) day⁻¹isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP⁺. In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.