Adapting Orthorep System for the Continuous Directed Evolution of Metabolic Plant Enzymes

Plant evolution has produced sub-optimal enzymes for our plant biotechnology requirements. By engineering of metabolic enzymes, it should be possible to alleviate constraints on plant metabolism currently imposed by kinetic properties or a short life of a protein. Directed evolution is a synthetic biology approach to engineer enzymes by harnessing the power of random mutagenesis and the screening of the desired characteristic in vitro or in vivo. “Continuous directed evolution” is a more efficient and scalable version that accomplishes the mutagenesis and selection step simultaneously in vivo, via error-prone replication of target gene and coupling of the microbial host´s growth rate to the target gene´s function. The orthogonal DNA replication (OrthoRep) is such a continuous directed evolution system with high mutagenicity (~10⁵ times that of the host genome) that allows the evolution of a target gene in yeast (Saccharomyces cerevisiae). It is based on a highly error-prone DNA polymerase that acts specifically on a linear plasmid-borne target gene without increasing the genomic mutation rate. As of now, OrthoRep has not been applied to engineer plant enzymes. Here, we present our pilot study to adapt OrthoRep to facilitate simple, high-throughput cloning of target plant genes. Yeast THI4 was chosen to test our modified OrthoRep system because, as a suicide enzyme, it requires a high level of expression to complement a thiazole auxotroph and thus represents a severe test of the expression system. The above pilot study indicates that our adapted OrthoRep system is in principle ready for the high-throughput evolution of metabolic enzymes from plants or other organisms.