Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction (dataset)

Carboxylic Acid Reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.

羧酸还原酶（Carboxylic Acid Reductases, CARs）是具备工业应用价值的生物催化剂。其固有特性——尤其是较差的结构稳定性——使其难以满足生物工业生产流水线的最优应用要求。本研究采用了新兴的工程化工具：祖先序列重构（Ancestral Sequence Reconstruction, ASR），该技术可在蛋白质全序列范围内筛选出兼具稳定作用与酶学中性的突变位点。我们采用三种算法结合的策略，对分枝杆菌属与诺卡氏菌属的CAR1直系同源物的功能性祖先序列完成了重构。经实验验证，祖先型羧酸还原酶（Ancestral CARs, AncCARs）属于CAR酶家族，且对芳香族羧酸类底物具有底物偏好性。祖先型酶对多种有机溶剂、pH环境以及模拟体内生理状态的盐浓度均展现出差异化的耐受性。相较于研究较为透彻的现存CAR酶，AncCARs的解链温度（Tm）最高可提升35℃，半衰期最长可达此前已报道最优水平的9倍。本研究通过祖先序列重构技术，为现有的CAR工具库新增了三款热稳定型羧酸还原酶，为实现高温条件下的醛类生物合成、拓展生物催化领域的全新应用场景提供了技术支撑。