设计改造羧酸还原酶合成医药中间体(S)-2-氨基丁醇  

Engineering of a carboxylic acid reductase-in the synthesis of pharmaceutical intermediate(S)-2-aminobutanol

作　　者：张晓辉 覃宗敏 李聪聪 路福平[1] 曲戈 孙周通 ZHANG Xiaohui;QIN Zongmin;LI Congcong;LU Fuping;QU Ge;SUN Zhoutong(School of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Innovation Center for Synthetic Biotechnology,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]国家合成生物技术创新中心,天津300308

出　　处：《生物学杂志》2024年第1期6-13,93,共9页Journal of Biology

基　　金：国家自然科学基金项目(31900909);中国科学院青年创新促进会项目(2021175);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJJ-005-08)。

摘　　要：(S)-2-氨基丁醇同时具有羟基及氨基官能团,是多种重要药物分子的关键手性中间体,生物合成(S)-2-氨基丁醇尚缺少有效的酶元件。以塞格尼氏菌(Segniliparus rugosus)来源的羧酸还原酶SrCAR为研究对象,通过对实验室已有SrCAR突变文库进行筛选测试,结合活性位点共进化分析,同时利用组合活性中心饱和突变策略(Combinatorial active-site saturation test,CAST)构建新的突变体文库,经测试最终获得优势突变体XH7(G430V/E533F/A627N)。该突变体催化底物N-Boc-(S)-2-氨基丁酸到醛产物的活性(kcat/Km)较野生型SrCAR提高2.1倍,热熔值(Tm)提升2.3℃。进一步通过引入荧光假单胞菌(Pseudomonas fluorescens)来源的醇脱氢酶PfADH,可还原N-Boc-(S)-2-氨基丁醛到醇产物。XH7和PfADH的双酶共表达体系反应5 h即可将20 mmol/L底物实现几乎完全转化,转化率达到99%,并经脱Boc保护与分离纯化获得终产物(S)-2-氨基丁醇,得率为60%。通过分子动力学模拟解析最优突变体活性及热稳定性提高的分子机制,为SrCAR酶设计改造提供新的研究思路,拓展酶法合成(S)-2氨基丁醇的生物酶工具箱,可为类似高附加值医药中间体的生物合成提供理论和实践指导。Armed with both hydroxyl and amino groups,(S)-2-aminobutanol is an essential chiral building block used in the preparation of diverse pharmaceutical drugs,while effective biocatalysts are highly desired toward the synthesis of(S)-2-aminobutanol.The carboxylic acid reductase derived from Segniliparus rugosus(SrCAR)was used as the model enzyme in the transformation of(S)-2-aminobutyric acid to(S)-2-aminobutanol combined with a heterologous alcohol dehydrogenase.After screening the previously constructed SrCAR mutant libraries,the active site was obtained and co-evolution analysis was performed.By using the combinatorial active-site saturation test strategy,the dominant mutant XH7(G430V/E533F/A627N)was obtained.The k cat/K m and T m values were improved 2.1 fold and 2.3℃compared to that of the wild type enzyme,respectively in the transformation of N-Boc-(S)-2-aminobutyric acid to N-Boc-(S)-2-aminobutyraldehyde.Further,N-Boc-(S)-2-aminobutyraldehyde to the alcohol product can be reduced by the introduction of Pseudomonas fluorescens(PfADH).The bi-enzymatic system containing of XH7 and PfADH enabled a 99%conversion toward 20 mmol/L substrate in 5 h.After deprotection and purification,the yield of(S)-2-aminobutanol reached 60%.Molecular dynamics simulations were performed to shed light on the molecular basis for the improved activity and thermostability of the best mutant.This study expands the biocatalytic toolbox for the synthesis of(S)-2-aminobutanol,and provides guidance for the biosynthesis of similar high value-added pharmaceutical intermediates.

关 键 词：羧酸还原酶 理性设计 生物催化 双酶级联 (S)-2-氨基丁醇 

分 类 号：Q55[生物学—生物化学] R91[医药卫生—药学]