Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
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DOI：
10.1038/s42003-019-0677-y
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发表时间：
2019-11
影响因子：
5.9
通讯作者：
Adam Thomas;R. Cutlan;W. Finnigan;M. van der Giezen;N. Harmer
中科院分区：
文献类型：
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作者：
Adam Thomas;R. Cutlan;W. Finnigan;M. van der Giezen;N. Harmer
羧酸还原酶（CAR）是具有工业重要性的生物催化剂。它们的特性，尤其是稳定性差，使其不适合在生物工业管道中使用。在这里，我们采用了祖先序列重建（ASR）——一种新兴的工程工具，可以识别整个蛋白质中稳定但酶中性的突变。我们使用三种算法方法来重建分枝杆菌和诺卡氏菌 CAR1 直系同源物的功能祖先。 Ancestral CAR（AncCAR）被证实是优先选择芳香族羧酸的 CAR 酶。祖先还表现出对溶剂、pH 值和体内盐浓度的不同耐受性。与经过充分研究的现有 CAR 相比，AncCAR 的 Tmup 高出 35°C，半衰期比之前观察到的最长半衰期长九倍。通过祖先重建，我们用三种新的热稳定 CAR 酶扩展了现有的 CAR 工具箱，提供了醛类高温生物合成的途径，以推动生物催化领域的新应用。
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 aTmup 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.