Identification of a novel ene reductase from Pichia angusta with potential application in ( R )-levodione production.

Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products. Herein, a novel FMN-binding ene reductase (PaER) from Pichia angusta was heterologously expressed in Escherichia coli BL21(DE3), and the recombinant enzyme was characterized for its biocatalytic properties. PaER displayed optimal activity at 40 °C and pH 7.5, respectively. The purified enzyme was quite stable below 30 °C over a broad pH range of 5.0-10.0. PaER was identified to have a good ability to reduce the C[double bond, length as m-dash]C bond of various α,β-unsaturated compounds in the presence of NADPH. In addition, PaER exhibited a high reduction rate ( k cat = 3.57 s -1 ) and an excellent stereoselectivity (>99%) for ketoisophorone. Engineered E. coli cells harboring PaER and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of ketoisophorone. As a result, up to 1000 mM ketoisophorone was completely and enantioselectively converted to ( R )-levodione with a >99% ee value in a space-time yield of 460.7 g L -1 d -1 . This study provides a great potential biocatalyst for practical synthesis of ( R )-levodione.