Department of Chemistry, Shanghai University, Shangda Road 99, Shanghai, China.
Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education and College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Appl Microbiol Biotechnol. 2023 Mar;107(5-6):1649-1661. doi: 10.1007/s00253-023-12389-4. Epub 2023 Jan 30.
(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 k/K 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.
(R)-4-丙基二氢呋喃-2(3H)-酮（R-PDFO）是抗癫痫药物布瓦西坦的关键手性中间体。由于缺乏一种简单且经济的方法来制备 R-PDFO，因此其生产也一直存在环保问题。在这里，我们使用烯还原酶从易于合成的 4-丙基呋喃-2(5H)-酮（PFO）开发了一种直接的生物还原方法。在筛选了 27 种烯还原酶后，E116 脱颖而出，起始酶的收率为 25.7%，ee 值为 97%（R）。为了提高 E116 的催化效率，首先进行了几轮定向进化。通过合理设计、丙氨酸扫描和随机诱变，获得了工程化的烯还原酶 E116-M3，其收率提高了 2.63 倍，k/K 提高了 12.6 倍，立体选择性提高到 99%（R）。为了进一步提高 R-PDFO 的产量，然后优化了反应条件。优化后的反应体系的催化活性再次提高了 2.3 倍，E116-M3 的周转数（TON）达到 705。随后，含有 E116-M3 的全细胞也显示出类似的合成 R-PDFO 的能力。最后，在 20mM PFO 负荷下，E116-M3 用于 50mL 规模的 R-PDFO 合成，实现了 81%的分离收率和 99%的 ee 值。总之，该工程烯还原酶催化 PFO 不对称还原的新方法可为高效合成 R-PDFO 提供一种绿色替代方案。 关键点： • 首次使用烯还原酶文库筛选 PFO 的生物还原。 • 合理设计有助于提高 PFO 还原的 R-立体选择性。 • E116-M3 具有高活性和对 R-PDFO 的立体选择性。
