Discovery, design, and engineering of enzymes based on molecular retrobiosynthesis

Biosynthesis—a process utilizing biological systems to synthesize chemical compounds—has emerged as a revolutionary solution to 21st‐century challenges due to its environmental sustainability, scalability, and high stereoselectivity and regioselectivity. Recent advancements in artificial intelligence (AI) are accelerating biosynthesis by enabling intelligent design, construction, and optimization of enzymatic reactions and biological systems. We first introduce the molecular retrosynthesis route planning in biochemical pathway design, including single‐step retrosynthesis algorithms and AI‐based chemical retrosynthesis route design tools. We highlight the advantages and challenges of large language models in addressing the sparsity of chemical data. Furthermore, we review enzyme discovery methods based on sequence and structure alignment techniques. Breakthroughs in AI‐based structural prediction methods are expected to significantly improve the accuracy of enzyme discovery. We also summarize methods for de novo enzyme generation for nonnatural or orphan reactions, focusing on AI‐based enzyme functional annotation and enzyme discovery techniques based on reaction or small molecule similarity. Turning to enzyme engineering, we discuss strategies to improve enzyme thermostability, solubility, and activity, as well as the applications of AI in these fields. The shift from traditional experiment‐driven models to data‐driven and computationally driven intelligent models is already underway. Finally, we present potential challenges and provide a perspective on future research directions. We envision expanded applications of biocatalysis in drug development, green chemistry, and complex molecule synthesis.