Directed evolution is typically a slow and labor-intensive process that relies on multiple cycles of mutation to accelerate the development of desired protein traits. Now, a platform, named T7-ORACLE, has been developed that enables fast and scalable protein evolution.

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Evolution is a fundamental process that describes changes in the genetic characteristics of life. However, natural selection alone only occasionally gives rise to organisms or biomolecules with desired traits. To achieve more frequent and targeted generation of beneficial phenotypes in proteins and enzymes, scientists developed directed evolution — a method that mimics Darwinian evolution in controlled laboratory settings1. Directed evolution revolutionized protein engineering by introducing beneficial mutations and selecting advantageous variants. However, traditional evolution methods, such as error-prone polymerase chain reaction, site saturation mutagenesis, deaminase-driven random mutation and recombination-based DNA shuffling, rely on iterative cycles of in vitro mutagenesis, transformation and selection, which are labor-intensive and limit both the depth and scale of evolutionary campaigns1,2. To overcome these constraints, continuous in vivo evolution systems, such as OrthoRep, BacORep and EcORep, have been developed that enable sustained mutagenesis of target genes3,4,5. Whereas systems such as OrthoRep in yeast suffer from slow generation times, BacORep and EcORep in bacteria are limited by low mutation rates (approximately 2.0 × 10−7 substitutions per base pair (spb)). Now, in an article published in Science, Diercks et al.6 report the development of T7-ORACLE (T7 orthogonal replisome-assisted continuous laboratory evolution), a method for protein evolution that achieves mutation rates of up to 1.7 × 10−5 spb in Escherichia coli and enables continuous hypermutation and accelerated evolution of the target gene.

Bacteriophage T7 owns a linear double-stranded genome that is replicated by a precisely regulated T7 replisome. Utilizing the unique DNA replication mechanism of the T7 replisome, Diercks et al.6 designed an orthogonal T7 replication module anchored to the T7 replisome and decoupled from the host’s chromosomal replication (Fig. 1). The designed T7-ORACLE system uses a defined set of T7 replication proteins and engineered T7 DNA polymerase variants to bias mutagenesis toward the T7 origin plasmid while minimizing crosstalk with the host genome. This orthogonality enables aggressive exploration of sequence mutations without causing lethal genomic instability. Specifically, the T7-ORACLE system was implemented in an E. coli strain in which five essential T7 replication proteins — T7 RNA polymerase, T7 single-stranded DNA-binding protein, T7 helicase–primase, T7 DNA polymerase and T7 lysozyme — are expressed. The replication of the T7 origin plasmid is initiated by T7 RNA polymerase to produce the T7 transcript, which serves as the primer of T7 DNA polymerase. The helicase–primase fusion protein is then recruited to the transcription bubble, resulting in concerted synthesis of both the leading and lagging strands. This process is further stabilized by the presence of the T7 single-stranded DNA-binding protein.

Fig. 1: The design of T7-ORACLE.

In T7-ORACLE, the designated gene inserted into a T7 origin plasmid is first transcribed by T7 RNA polymerase in conjunction with T7 lysozyme to generate a T7 RNA primer. Mutation of the target gene is achieved by the T7 replisome. The mutated T7 DNA polymerase is primed by an orthogonal transcript, and T7 helicase and primase are recruited to the transcription bubble, enabling concerted leading- and lagging-strand synthesis. The T7 single-stranded DNA-binding protein stabilizes the replication loop.

A key innovation involved the fusion of a hydrolase-deficient T7 lysozyme to T7 RNA polymerase to stimulate the replication initiation. This platform supports stable plasmid replication with a transformation efficiency of 2.4 × 1010 CFU μg−1, orders of magnitude higher than those of linear orthogonal replicons5,7. Central to the system’s function is the engineered mutagenicity of T7 DNA polymerase. Starting with an exonuclease-deficient variant of T7 DNA polymerase, the team used directed evolution to introduce mutations that reduce base-selection fidelity. Through saturation mutagenesis and screening, they identified key mutations that collectively raised the mutation rate of the target gene to 1.7 × 10−5 spb — a 100,000-fold increase over the mutation rate of the E. coli genome.

To demonstrate the utility of the T7-ORACLE platform for protein evolution, Diercks et al.6 applied it to the continuous evolution of TEM-1β-lactamase. Using the T7 replisome with a selected T7 DNA polymerase variant, they obtained TEM-1 variants that showed a 5,000-fold increase in activity against monobactam and cephalosporin antibiotics within just 6 days. Deep sequencing of evolved populations uncovered mutational trajectories that recapitulated known clinical resistance pathways, including key amino acid substitutions and promoter mutations that boost expression.

A compelling feature of T7-ORACLE is its compatibility with prediversified libraries. When a site-saturation library targeting five active-site residues of TEM-1 was used as the starting point under cefotaxime selection, evolution yielded diverse solutions distinct from those originating from the wild-type gene, highlighting T7-ORACLE for exploring broader fitness landscapes and escaping local optima. Owing to the homogeneous characteristics of the T7 DNA polymerase mutant, the T7-ORACLE system exhibits uniform distribution of target mutation sites without discernible bias.

Overall, this study developed an orthogonal replication system for continuous hypermutation in E. coli based on the replisome of bacteriophage T7. The core advantage of the T7-ORACLE system lies in confining mutagenesis to the plasmid, thereby preserving the host’s genome integrity. Compared with OrthoRep and EcORep, T7-ORACLE combines extreme mutagenesis rates with the practical advantages of circular plasmid replication, offering high transformation efficiency and ease of library construction. Moreover, in contrast to phage-assisted continuous evolution8, which relies on specialized continuous culture equipment, T7-ORACLE functions effectively in standard batch cultures, making it widely accessible to laboratories with basic molecular biology capabilities.

The system currently lacks inducible control over replisome expression. Introducing inducible switches to regulate replication activity in response to external cues could offer precise temporal control, facilitating phase-specific evolution and more refined management of selective pressure. Although the construction of a genome-integrated replisome simplifies adoption, further refinements are needed to improve versatility and address challenges such as temporal mutation rate control. Whereas T7-ORACLE is currently primarily used for the evolution of resistance proteins, its application to other functional proteins remains challenging. Moreover, integrating T7-ORACLE with machine learning-guided design could considerably enhance efficiency by predicting productive mutational trajectories and prioritizing informative libraries, thereby reducing the experimental screening efforts9,10.

In summary, the work by Diercks et al.6 established a powerful orthogonal replication system for continuous hypermutation and accelerated evolution in E. coli. T7-ORACLE opens new avenues for protein engineering and evolutionary studies. Benefiting from its rapid mutagenesis, accelerated evolution rate and high compatibility, T7-ORACLE holds great potential for broad applications in protein engineering. It can be used to develop proteins with desirable traits, such as highly selective and high-affinity antibodies and enzymes with novel specificities or enhanced catalytic activities. Furthermore, it may serve as a valuable tool for investigating the mechanisms behind the emergence of drug-resistant mutations in therapeutic targets.

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Authors and Affiliations

Department of Occupational and Environmental Health, School of Public Health, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, State Key Laboratory of Metabolism and Regulation in Complex Organisms, Wuhan University, Wuhan, China

Jun Xiong, Neng-Bin Xie & Bi-Feng Yuan

Authors

1. Jun Xiong
2. Neng-Bin Xie
3. Bi-Feng Yuan

Corresponding author

Correspondence to Bi-Feng Yuan.

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The authors declare no competing interests.

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Xiong, J., Xie, NB. & Yuan, BF. A super protein evolution engine. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02065-1

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Published: 04 November 2025

Version of record: 04 November 2025

DOI: https://doi.org/10.1038/s41589-025-02065-1