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Engineering of an ene-reductase for producing the key intermediate of antiepileptic drug Brivaracetam.
- Source :
-
Applied Microbiology & Biotechnology . Mar2023, Vol. 107 Issue 5/6, p1649-1661. 13p. - Publication Year :
- 2023
-
Abstract
- (R)-4-Propyldihydrofuran-2(3H)-one (R-PDFO) is the key chiral intermediate for the antiepileptic drug Brivaracetam. Lacking a simple and economical method to approaching R-PDFO, the production of R-PDFO also remains environmentally unfriendly. Here, we developed a straightforward bioreduction way from easily synthesized 4-propylfuran-2(5H)-one (PFO) using ene-reductases. After screened with 27 ene-reductases, E116 stood out with 25.7% yield and 97% ee (R) as the starting enzyme. To improve the catalytic efficiency of E116, several rounds of directed evolution were first carried out. Through rational design, alanine scanning and random mutagenesis, engineered ene-reductase E116-M3 was obtained, with a 2.63-fold improvement in yields over WT, a 12.6-fold improvement in kcat/Km over WT, and stereoselectivity increased to 99% (R). To further improve the yield of R-PDFO, the reaction conditions were then optimized. The catalytic activity of the optimized reaction system was increased again by 2.3 times and the turnover number (TON) of E116-M3 reached 705. Subsequently, whole cells harboring E116-M3 were also shown to have similar capabilities of synthesizing R-PDFO. Finally, E116-M3 was employed in the 50-mL-scale synthesis of R-PDFO under 20 mM of PFO loading to achieve 81% isolated yield and 99% ee. In conclusion, this new approach of engineered ene-reductase catalyzing the asymmetric reduction of PFO could be a green alternative for the efficient synthesis of R-PDFO. Key points: • An ene-reductase library was first used to screen the bioreduction of PFO. • Rational design contributed to the enhanced R-stereoselectivity of PFO reduction. • E116-M3 was obtained with high activity and stereoselectivity for R-PDFO. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01757598
- Volume :
- 107
- Issue :
- 5/6
- Database :
- Academic Search Index
- Journal :
- Applied Microbiology & Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 162358529
- Full Text :
- https://doi.org/10.1007/s00253-023-12389-4