Identification of a novel ene reductase from with potential application in ()-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 was heterologously expressed in 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 ( = 3.57 s) and an excellent stereoselectivity (>99%) for ketoisophorone. Engineered 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 ()-levodione with a >99% ee value in a space-time yield of 460.7 g L d. This study provides a great potential biocatalyst for practical synthesis of ()-levodione.