Inducible directed evolution of complex phenotypes in bacteria

Abstract Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5–10 kb in length, elimination o...

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Bibliographic Details
Published in:Nucleic acids research 2022-06, Vol.50 (10), p.e58-e58
Main Authors: Al’Abri, Ibrahim S, Haller, Daniel J, Li, Zidan, Crook, Nathan
Format: Article
Language:English
Subjects:
Bacteria - genetics
Bacteria - metabolism
Bacteria - virology
Bacteriophages - genetics
Directed Molecular Evolution - methods
Methods Online
Mutagenesis
Phenotype
Plasmids - genetics
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Summary:Abstract Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5–10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli’s utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkac094