Two-Enzyme Coexpressed Recombinant Strain for Asymmetric Synthesis of Ethyl (R)-2-Hydroxy-4-phenylbutyrate

Abstract

( R )-2-Hydroxy-4-phenylbutyrate (HPBE) is an important chiral intermediate for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Asymmetric reduction of ethyl 2-oxo-4-phenyl-butyrate (OPBE) to ( R )-HPBE using a recombinant strain can provide high enantioselectivity. Cofactor regeneration is a critical issue in the application of a recombinant strain. A carbonyl reductase gene ( iolS ) and a glucose dehydrogenase (GDH) gene from Bacillus subtilis were cloned. Recombinant IolS was purified using a Ni-NTA column and its enzyme activity properties were investigated. The purified IolS exhibited maximum activity at pH 6.0 and 30°C, and the enzyme showed good thermostability below 40 °C. It retained over 75% of its activity in the acidic pH range of 5.5(7.0. Three coexpression strategies were used for the recombinant vectors. The recombinant E. coli strain containing polycistronic plasmid pET-G-T7-I showed excellent carbonyl reductase activity, and the specific activity of both IolS and GDH in the crude cell extract reached 1.5 U/mg. In the asymmetric reduction of OPBE by recombinant E. coli cells in aqueous system, the yield of ( R )-HPBE reached over 99% with an enantiomeric excess of 99.5% at 10 g/L of OPBE within 15 h. 克隆了来自于枯草芽孢杆菌的羰基还原酶基因 IolS 和葡萄糖脱氢酶基因 GDH, 采用 Ni-NTA 镍亲和层析柱对重组蛋白 IolS 进行纯化, 并对纯酶进行了酶学性质研究. 结果表明, 该羰基还原酶的最适温度和 pH 值分别为 30 °C 和 6.0; 在 40 °C 以下具有较好的热稳定性; 在 pH 5.5-7.0 的偏酸性范围内能保持 75% 以上的酶活. 采用三种策略构建了 IolS 和 GDH 的共表达重组质粒, 结果发现, 采用双启动子的重组质粒能够实现羰基还原酶 IolS 的高效表达, 粗酶液中的 IolS 和 GDH 的比酶活均达到 1.5 U/mg. 运用该重组菌对 10 g/L 的 OPBE 进行不对称还原, 反应 15 h 后, 底物转化率大于 99%, 产物 ( R )-2-羟基-4-苯基丁酸乙酯的 ee 值达到 99.5%. A carbonyl reductase (IolS) exhibiting high enantioselectivity in the reduction of OPBE to ( R )-HPBE was cloned, characterized, and coexpressed with glucose dehydrogenase to construct recombinant E. coli with cofactor regeneration.