Beyond enzyme engineering: ordered enzyme immobilization drives enhanced productivity in vitro.

Increasing environmental and economic pressures associated with global fossil fuel demand necessitate a shift toward sustainable fuel production. Production of second-generation biofuels, such as isobutanol, presents a promising opportunity; however, product toxicity limits in vivo production, motivating the development of optimized in vitro systems. As prior efforts have focused on enzyme engineering to improve titer, these systems remain constrained by diffusion-limited mass transfer. Here, we introduce an engineered, ordered cellulosome-based immobilization system that facilitates enhanced enzymatic productivity. Using keto-acid decarboxylase, alcohol dehydrogenase, and formate dehydrogenase as a model for the final steps of the isobutanol pathway, the system achieved a preliminary isobutanol titer of 5.92 g/L, a 78.4% yield, and an enzymatic productivity of 0.34 mL-1 h-1, representing marked improvement over previous cell-free approaches. This proof-of-concept approach introduces the importance of targeted immobilization alongside enzyme optimization and demonstrates ordered scaffoldin-mediated immobilization as a versatile platform approach for future in vitro biofuel and biochemical production.