Semi-automated biofoundry workflows for sequence coevolution-guided isoprene synthase engineering.

Biofoundries serve as transformative platforms for accelerating the engineering of enzymes and microorganisms toward biomanufacturing. In this study, we developed scalable enzyme engineering workflows tailored for biofoundry applications, focusing on isoprene synthase (IspS) - a critical rate-limiting enzyme in the isoprene biosynthesis. By integrating computational mutation design based on sequence coevolution analysis and laboratory automation, we conducted three rounds of site-directed mutagenesis and screening. Approximately 100 genetic mutants were synthesized per round and these workflows can be easily scaled up to thousands without extensive optimization. Moreover, this approach enabled the rapid identification of IspS variants with up to 4.5-fold improvement in catalytic efficiency and simultaneously enhanced thermostability. Additionally, introducing the engineered IspS into Methylococcus capsulatus Bath improved methane-to-isoprene bioconversion, achieving a titer of 319.6 mg/l. These scalable workflows establish a robust framework for enzyme engineering within biofoundries. This provides a basis for the development of innovative biotechnological advancements.