Hydrogel-Immobilized Multienzyme Systems for Cell-Free Chemical Bioproduction

In this paper, Sugianto, et al. in the Carothers Lab demonstrated the embedding of enzymes expressed in myTXTL into hydrogels. The enzymes encompassed a multistep pathway for the production of malic acid from serine substrate. Four enzymes in this process were co-expressed in a single myTXTL reaction, allowing the rapid generation of multiple proteins and their direct transfer into the hydrogel for immobilization. Embedded enzymes were then assessed for activity and the ability to make malic acid, a rapid workflow for testing and optimization of enzyme pathways not possible in vivo or when relying on purified proteins. Additionally, DNA template concentration or promoter activity can be tuned for optimization of metabolic enzyme expression for each step in the pathway.