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 degrees C and pH 7.5, respectively. The purified enzyme was quite stable below 30 degrees C over a broad pH range of 5.0-10.0. PaeR was identified to have a good ability to reduce the C=C bond of various alpha,beta-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 biocataIysts 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 biocataIyst for practical synthesis of (R)-levodione.

Automated summary

This study reports the heterologous expression of a novel FMN-binding ene reductase (PaER) from Pichia angusta and its excellent catalytic performance in the asymmetric reduction reaction, especially for the selective reduction of ketoisophorone. Through the engineering of E. coli expression system, efficient asymmetric reduction of ketoisophorone was achieved.