Co-Immobilization of SpyTag-Cyclized Enzymes on a γPFD-SpyCatcher Hydrogel to Address Broad Specificity

The broad substrate specificity of enzymes, while advantageous for catalytic diversity, often leads to undesired side reactions and reduced product yields in engineered metabolic pathways. To address this challenge, we developed a programmable protein scaffold based on a self-assembled γPFD-SpyCatcher hydrogel for the in vivo co-immobilization of SpyTag-cyclized cascade enzymes, enabling the co-immobilization of cascade enzymes in a spatially organized manner. Enzymes with broad substrate specificities were linearly fused with SpyTags, facilitating their spatial organization on the nanoscaffold within engineered E. coli to ensure directed catalytic flux. Using this strategy, the yields of pinene and caffeoyl-CoA were enhanced by 5.8-fold (reaching 94.5 mg/L) and 2.4-fold (reaching 78.6 mg/L), respectively, compared to free enzyme systems. This work establishes an effective approach to mitigate the limitations posed by broad enzyme specificity and demonstrates its potential for applications in synthetic biology and industrial biotechnology.