Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.

BACKGROUND: Classical directed evolution is a powerful approach for engineering biomolecules with improved or novel functions. However, it traditionally relies on labour- and time-intensive iterative cycles, due in part to the need for multiple molecular biology steps, including DNA transformation, and limited screening throughput. RESULTS: In this study, we present an ultrahigh throughput in vivo continuous directed evolution system with thermosensitive inducible tunability, which is based on error-prone DNA polymerase expression modulated by engineered thermal-responsive repressor cI857, and genomic MutS mutant with temperature-sensitive defect for fixation of mutations in Escherichia coli. We demonstrated the success of the in vivo evolution platform with β-lactamase as a model, with an approximately 600-fold increase in the targeted mutation rate. Furthermore, the platform was combined with ultrahigh-throughput screening methods and employed to evolve α-amylase and the resveratrol biosynthetic pathway. After iterative rounds of enrichment, a mutant with a 48.3% improvement in α-amylase activity was identified via microfluidic droplet screening. In addition, when coupled with an in vivo biosensor in the resveratrol biosynthetic pathway, a variant with 1.7-fold higher resveratrol production was selected by fluorescence-activated cell sorting. CONCLUSIONS: In this study, thermal-responsive targeted mutagenesis coupled with ultrahigh-throughput screening was developed for the rapid evolution of enzymes and biosynthetic pathways.