基于单细胞超高通量筛选的转氨酶B3-TA分子改造

ω-transaminases are efficient biocatalysts for the asymmetric synthesis of chiral amine compounds. However, the lack of high throughput screening methods is the bottleneck for the molecular engineering of transaminases. In our previous studies, we established a droplet-microfluidic-based single-cell ultrahigh throughput screening system, and successfully applied it for the directed evolution of an esterase with improved stereoselectivity. On this basis, the project intends to develop a fluorescence coupling strategy for determining the catalytic activity of ω-transaminases in microfluidic droplets. Further, this project will establish a universal single-cell ultrahigh throughput screening system for ω-transaminases. This screening system will dramatically increase the screening throughput of ω-transaminases from less than 10,000 per day of the traditional 96-well plate assays to more than 10 million per day, thus greatly improving the efficiency of molecular evolution of ω-transaminases. In this project, the key intermediate in the synthesis of levocetirizine, (R)-4-chlorodiphenylammonia, will be used as a model for the directed evolution of a thermophilic transaminase B3-TA with improved catalytic efficiency. Finally, the molecular mechanism of the best mutants will be studied. The ultrahigh throughput screening system established in this project can be applied to a variety of industrial valuable ω-transaminases and is expected to break through the bottleneck of the molecular evolution of ω-transaminases, thus promoting the development of high-quality ω-transaminases resources.

ω-转氨酶能高效催化手性胺化合物的不对称合成，利用蛋白质工程技术改善天然转氨酶底物谱窄、活性低、稳定性差等缺点已经成为学术界关注的热点，但现有筛选方法较低的通量是限制转氨酶分子改造的瓶颈。本项目拟以转氨酶B3-TA催化合成重要医药中间体(R)-4-氯二苯甲氨为模式，建立基于“荧光激活微液滴分选”的单细胞超高通量ω-转氨酶筛选系统。通过构建转氨酶活性的荧光偶联策略，在微液滴中将酶活性转化为荧光信号，并利用微流控检测/分选芯片在单细胞层面对酶活性进行定量分析及高速分选。该筛选系统能够将转氨酶的筛选通量从现有微孔板法的每天不超过1万个提高到每天1000万个以上，且广泛适用于各种ω-转氨酶，有望打通转氨酶分子改造的瓶颈，为酶学理论及绿色生物催化领域的进步提供技术支撑。

ω-转氨酶能高效催化手性胺化合物的不对称合成，利用蛋白质工程技术改善天然转氨酶底物谱窄、活性低、稳定性差等缺点是学术界关注的热点，但传统筛选方法较低的通量是限制转氨酶分子改造的瓶颈。我们在前期研究中，建立了基于液滴微流控的单细胞超高通量筛选系统，并成功地将其应用于酯酶的定向进化，使其具有更高的立体选择性。本研究我们在微流控液滴内开发了一种新型转氨酶单细胞荧光偶联方法，并以此为基础建立了基于“荧光激活微液滴分选”的单细胞超高通量转氨酶筛选体系，筛选通量高达每天1000万个突变体，具有良好的通用性，能够满足大多数转氨酶的多种性质的筛选。基于该筛选系统，本研究实现了对转氨酶的定向进化，筛选出对大底物4-氯二苯甲酮具有高催化活性（活性提升10倍以上）的突变体4个，其中最优突变体（突变位点为F89A/Y154F/C417A/P419H）的催化活性提升高达23.5倍；此外，解析了突变位点影响转氨酶催化效率的分子机制：转氨酶的底物结合口袋空间位阻减小并且与底物4-氯二苯甲酮增加了氢键相互作用，结合能力更强。最终为重要医药中间体——左旋西替利嗪的生物催化合成以及产业化提供了极具应用潜力的新酶资源。