Identification of a novel ene reductase from Pichia angusta with potential application in (R)-levodione production. Issue 22 (10th May 2022)

• Record Type: Journal Article
• Authors: Zhang, Baoqi; Sun, Jiale; Zheng, Yanqiu; Mao, Xinlei; Lin, Jinping; Wei, Dongzhi
• Is Part Of: RSC advances. Volume 12:Issue 22(2022)
• Page Start: 13924
• Page End: 13931
• Publication Date: 2022-05-10
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2ra01716d
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products. Abstract : 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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.