An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.
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| Title: | An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli. |
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| Authors: | Diercks, Christian S., Sondermann, Philipp, Rong, Cynthia, Gillis, Thomas G., Ban, Yahui, Wang, Celine, Dik, David A., Schultz, Peter G. |
| Source: | Science. 8/7/2025, Vol. 389 Issue 6760, p618-622. 5p. |
| Subjects: | Replisomes, DNA replication, Genomes, Bacteriophages, Drug resistance in bacteria |
| Abstract: | Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10−5 substitutions per base in vivo—100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week. Editor's summary: Continuous evolution of proteins in the lab is often slow because normal mutation rates in bacteria are very low. Diercks et al. investigated whether a highly mutagenic DNA replication system could speed up evolution in Escherichia coli without harming the host genome. They engineered an orthogonal T7 replisome that replicates only target plasmids at mutation rates 100,000 times higher than normal while leaving the rest of the genome unchanged. Using this system, the authors rapidly evolved TEM-1 β-lactamase to gain much stronger resistance to several antibiotics in under a week. This approach could greatly accelerate protein engineering and antibiotic resistance studies. —Di Jiang [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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| Abstract: | Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10−5 substitutions per base in vivo—100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week. Editor's summary: Continuous evolution of proteins in the lab is often slow because normal mutation rates in bacteria are very low. Diercks et al. investigated whether a highly mutagenic DNA replication system could speed up evolution in Escherichia coli without harming the host genome. They engineered an orthogonal T7 replisome that replicates only target plasmids at mutation rates 100,000 times higher than normal while leaving the rest of the genome unchanged. Using this system, the authors rapidly evolved TEM-1 β-lactamase to gain much stronger resistance to several antibiotics in under a week. This approach could greatly accelerate protein engineering and antibiotic resistance studies. —Di Jiang [ABSTRACT FROM AUTHOR] |
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| ISSN: | 00368075 |
| DOI: | 10.1126/science.adp9583 |
