Harnessing ancestral sequence reconstruction for engineering novel and robust enzymes

Biocatalysis has emerged as an essential green manufacturing approach, enabling sustainable and efficient synthesis across the pharmaceutical, agricultural, and food industries. Beyond its fundamental significance in elucidating molecular evolution, ancestral sequence reconstruction (ASR) has also proven to be a powerful technology for engineering enzymes with enhanced properties or novel functionalities. ASR infers the sequences of ancestors by comparing the sequences of extant (current) enzymes. This approach has proven highly versatile, not only facilitating the generation of robust enzymes with increased activity and/or promiscuity, improved stability, and selectivity but also offering a unique framework for investigating fundamental mechanisms and biochemical principles that are difficult to address using contemporary enzymes alone. This review highlights the advances in ASR over the past three years across diverse applications, including regulation of enzyme function and selectivity, investigation of functional divergence mechanisms, enhancement of thermostability and catalytic activity, and expansion of substrate scope. We anticipate that ASR will play an increasingly central role in advancing biocatalytic applications. Indeed, the field of enzyme engineering is currently undergoing a transformative phase, driven by the synergistic integration of computational evolutionary tools and experimental validation, which continues to unlock the functional potential of biocatalysts.