Viral Enzyme Accelerates Evolution of Therapeutic Proteins

In nature, evolution takes years to sculpt new traits. In the lab, scientists hope to achieve this feat much faster: Being able to evolve proteins with enhanced or novel functions has important applications, including engineering antibodies or enzymes to treat diseases.

In a recent study published in Science, researchers led by Scripps Research biochemist Peter Schultz developed a synthetic biology framework that accelerated the rate of evolution, producing protein variants within days.¹ Their findings lay the groundwork to engineer therapeutic proteins against diseases including cancers and neurodegeneration.

“This is like giving evolution a fast-forward button,” said Schultz in a press release. “You can now evolve proteins continuously and precisely inside cells without damaging the cell’s genome or requiring labor-intensive steps.”

To evolve proteins with desired functions, scientists have been using an approach called directed evolution, which mimics natural selection. This involves using orthogonal replication systems that operate independently from the host cell’s machinery. These error-prone replication tools introduce mutations in genes expressed within host cells like Escherichia coli or yeast, enabling researchers to select variants with the required traits.^2,3 However, the existing approaches are time-consuming and show modest mutation rates.

In the present study, Schultz and his team circumvented this challenge by utilizing the replication machinery, or replisome, of the T7 bacteriophage, a virus that infects most E. coli strains. They introduced plasmids carrying genes encoding the T7 replisome into E. coli cells, which led to mutations in a target gene expressed in the bacterium.

Engineering a T7 replication enzyme deficient in repair mechanisms made the system more error-prone, enabling the researchers to introduce mutations at a much higher rate than the original T7 replication system without damaging the host cells. The researchers dubbed this system T7 orthogonal replisome-assisted continuous laboratory evolution (T7-ORACLE).

Finally, Schultz and his team tested T7-ORACLE’s utility for evolving proteins. They focused on β-lactamase, a bacterial enzyme that confers resistance to β-lactam antibiotics like penicillin and cephalosporins. They introduced plasmids carrying T7-ORACLE and a gene encoding β-lactamase into E. coli cells and exposed these bacteria to increasing concentrations of various antibiotics for six days. Within this time, the bacteria grew resistant to antibiotic levels up to 5,000 times higher than the initial dose.

Comparing the sequence of antibiotic resistance genes before and after antibiotic treatment revealed that the genes accumulated several mutations. To better understand the molecular basis for resistance, Schultz and his team sequenced the antibiotic resistance genes after each escalating antibiotic dosage. This helped them identify the timeline of mutations that occurred first, followed by additional mutations that accumulated and helped the bacterium thrive. A majority of the mutations they observed had been reported in clinical isolates or laboratory evolution experiments.^4,5

“The surprising part was how closely the mutations we saw matched real-world resistance mutations found in clinical settings,” said study coauthor Christian Diercks, a chemist at Scripps Research.

While the researchers tested their proof of concept using one gene encoding an antibiotic resistance-associated enzyme, in theory, scientists could evolve any other protein to yield variants with desired functions, including to treat diseases. “What’s exciting is that it’s not limited to one disease or one kind of protein,” said Diercks. “Because the system is customizable, you can drop in any gene and evolve it toward whatever function you need.”