ArticleMicrobial biotechnology2021
Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast.
Article in Microbial biotechnology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 40 citations in OpenAlex.
- Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter.Molecular systems biology · 2025Article
- Ultra-efficient Integration of Gene Libraries onto Yeast Cytosolic Plasmids.ACS synthetic biology · 2025Article
- Recent advances in targeted mutagenesis to expedite the evolution of biological systems.Journal of microbiology (Seoul, Korea) · 2025Review
- Genetically encoded biosensors for the circular plastics bioeconomy.Metabolic engineering communications · 2024Article
- Continuous evolution of user-defined genes at 1 million times the genomic mutation rate.Science (New York, N.Y.) · 2024Article
- MutaT7ACS synthetic biology · 2024Article
- Catalytic carbon-carbon bond cleavage in lignin via manganese-zirconium-mediated autoxidation.Nature communications · 2024Article
- Evolution and synthetic biology.Current opinion in microbiology · 2023Review
- Adapting enzymes to improve their functionality in plants: why and how.Biochemical Society transactions · 2023Review
- Engineering of Synthetic Transcriptional Switches in Yeast.Life (Basel, Switzerland) · 2022Review
- Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering.ACS synthetic biology · 2022Article
- In vivo hypermutation and continuous evolution.Nature reviews. Methods primers · 2022Article
- Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast.Microbial biotechnology · 2021Article
- Evolving Small-Molecule Biosensors with Improved Performance and Reprogrammed Ligand Preference Using OrthoRep.ACS synthetic biology · 2021Article
Corrections and comments
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Authors and funding
7 authors at 3 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Directed 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.
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