Directed Coevolution of Two Cellulosic Enzymes in Escherichia coli Based on Their Synergistic Reactions

Directed evolution is a widely used technique for improving enzymatic properties. The development of an efficient high-throughput screening method is a key procedure, which is however often unavailable for many enzyme reactions, including the cellulase-catalyzed cellulose hydrolysis. Here, we describe a high-throughput screening assay for directed coevolution of two cellulases (an endoglucanase and a β-glucosidase) in form of a bicistronic operon based on their synergistic reactions. Insoluble filter paper is used as the real cellulose substrate to screen for positive enzyme variants, facilitated by the colorimetric assay coupled to glucose liberated from cellulose under catalysis of endoglucanase and β-glucosidase. Directed coevolution saves the labor and time required for two independent directed evolution cycles, which might provide reference for the engineering of other cellulosic enzymes or multienzyme systems.