Design and validation of an inducible and curable EvolvR system for directed evolution in Corynebacterium glutamicum

The EvolvR system was developed as a tool for the continuous diversification of user-defined genomic loci in Escherichia coli . To make EvolvR accessible for enzyme engineering in Corynebacterium glutamicum , this work focused on the design and construction of gpEvolvR — a modified plasmid active in both Escherichia coli and Corynebacterium glutamicum with additional recombinant features enabling its complete curing after mutagenesis. First, the pSC101 origin of replication of gpEvolvR itself served as the proof-of-principle mutagenesis target. Two examined E. coli mutants exhibited substantial increases in kanamycin minimal inhibitory concentrations of +44% and +96% and thus confirmed the functionality of the new gpEvolvR plasmid system. Secondly, the EvolvR-based directed evolution of a genomically located gene was shown in C. glutamicum . Given that the β-carotene ketolase (CrtW) has been identified as a key optimization target in previous metabolic engineering studies of C. glutamicum , EvolvR was successfully applied to generate mutants with varying activities, which were identified through plate-based screening. Although none of the examined crtW mutants outperformed the reference sequence in production experiments, this study introduced the newly constructed and curable EvolvR system as a tool for targeted hypermutation in C. glutamicum .