Xiaoyu Chen,Zhe Dou,Tianwei Luo,Zewen Sun,Hongmin Ma,Guochao Xu,Ye Ni
Ancestral enzymes are promising for industrial biotechnology due to high stability and catalytic promiscuity. An effective protocol was developed for the directed resurrection of ancestral enzymes. Employing genome mining with diaryl alcohol dehydrogenase Kp ADH as the probe, descendant enzymes D10 and D11 were firstly identified. Then through ancestral sequence reconstruction, A64 was resurrected with a specific activity of 4.3 U·mg −1 . The optimum pH of A64 was 7.5, distinct from 5.5 of D10. The T15 50 and T m values of A64 were 57.5 °C and 61.7 °C, significantly higher than those of the descendant counterpart. Substrate spectrum of A64 was quantitively characterized with a Shannon-Wiener index of 2.38, more expanded than D10, especially, towards bulky ketones in Group A and B. A64 also exhibited higher enantioselectivity. This study provides an effective protocol for constructing of ancestral enzymes and an efficient ancestral enzyme of industrial relevance for asymmetric synthesis of chiral alcohols.
Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C–H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.
危险品化学品经营许可证（带存储）