Establishing a Serine Integrase‐Based Genetic Memory System In Vitro

The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high-density storage medium, offers a promising solution for creating genetic memory systems that can provide state-dependent responses to various stimuli. To date, numerous studies have reported on genetic memory systems in living organisms. However, developing modular, orthogonal, and quantifiable in vitro genetic memory systems with scalable biological components remains a significant challenge. In this study, we present an in vitro genetic memory system utilizing three orthogonal serine integrases for DNA-based information storage and processing. By organizing the system into three standardized modules featuring two noncovalent chemical interactions (streptavidin-biotin and parS-ParB), we successfully designed and tested the orthogonality, scalability, and functionalization of these systems. Notably, we expanded the application to implement a cascade biotransformation process converting styrene to (S)-1-phenyl-1,2-ethanediol ((S)-PED) with remarkable efficiency, achieving up to double the transformation rate compared to free-floating purified enzymes. We anticipate that these constructions hold significant potential for advancing artificial memory systems in vitro and will provide a reliable framework for the development of programmable biochemical functions in synthetic biology.