SummaryDirected evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces is a labour‐intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high‐throughput using a transcription factor‐based biosensor. We demonstrate the strengths of this workflow by evolving a rate‐limiting enzymatic reaction of the biosynthetic pathway for cis,cis‐muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high‐throughput sorting of best‐performing variants using a transcription factor‐based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13‐fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

SummaryDirected evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection, and amplification. While powerful, systematic searches through large sequence-spaces is a labor-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generates enzyme variants using the recently developed orthogonal replication system, OrthoRep, and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a ratelimiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers >13-fold compared to reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study, is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.