Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo.

Abstract:

:Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per day, rivalling the rate in typical in vitro mutagenesis for directed evolution of proteins, and promotes rapid continuous evolution of model proteins for antibiotic resistance and allosteric activation. eMutaT7 provides a very simple and tunable method for continuous directed evolution of proteins, and suggests that the fusion of new DNA-modifying enzymes to the orthogonal RNA polymerase is a promising strategy to explore the expanded sequence space without compromising gene specificity.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Park H,Kim S

doi

10.1093/nar/gkaa1231

subject

Has Abstract

pub_date

2021-01-06 00:00:00

eissn

0305-1048

issn

1362-4962

pii

6066637

pub_type

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