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Biological Chemistry

Directed Evolution Keeps Pace

Chemical Biology: Stringency modulation and negative selection in phage-assisted continuous evolution lead to better outcomes

by Celia Henry Arnaud
February 10, 2014 | A version of this story appeared in Volume 92, Issue 6

RNA polymerase that binds to a new promoter after phage-assisted continuous evolution.
Credit: Nat. Chem. Biol.
Liu and coworkers have developed an improved form of PACE that evolves an RNA polymerase with highly altered promoter specificity. Some of the revised enzyme’s mutations (blue) enable recognition of the new promoter. Others evolve during (red) or after (magenta) negative selection to discriminate against the original promoter.

A technique that enables proteins to be evolved in the lab in a continuous manner without user intervention has proven useful in creating proteins with new properties. Now, the researchers who devised the technique have added capabilities that expand the method’s utility.

Phage-assisted continuous evolution (PACE), first developed in 2011 by David R. Liu and coworkers at Harvard University, harnesses the rapid life cycle of bacteria-infecting viruses called bacteriophages to enable rapid and continuous protein evolution. In the technique, genes for the proteins to be evolved are engineered into phages. Then, they are optimized as the phages undergo cycles of selective replication in a continuously flowing stream of host bacteria cells.

These phages lack the gene for a protein called pIII, which phages need to replicate. The technique ensures that host bacteria supply pIII only to phages that express a protein with desired activity.

Now, Liu and coworkers have altered PACE selection conditions in two ways, enabling the method to evolve enzymes with greatly altered specificities that weren’t previously accessible (Nat. Chem. Biol. 2014, DOI: 10.1038/nchembio.1453).

One improvement is a negative selection that discriminates against evolving proteins with undesired activities. The second makes it possible to precisely raise or lower the bar for phage survival. It uses small molecules to reduce the stringency of positive and negative selection when needed.

To achieve negative selection, the researchers identified a form of pIII that impedes phage production. They demonstrated that linking undesired activities to the production of this “pIII-neg” protein enables PACE to weed out proteins with unwanted attributes.

To modulate the selection stringency, Liu and coworkers added to the host bacteria a plasmid that produces precisely controlled amounts of pIII when a small molecule is added to the evolution stream. Adding more of this small molecule allows phages to generate infectious progeny even when an evolving protein’s activity isn’t high enough to make sufficient pIII to ensure phage propagation.

In 2011, Liu and coworkers used the original PACE technique to generate an RNA polymerase that binds a promoter different from that bound by the native version. The effort required two evolution experiments linked by a hybrid promoter “stepping stone.” With the two improvements, they find that in a single experiment they can evolve highly active RNA polymerases that recognize new target promoters and reject their original promoters.

The advances have made PACE “significantly better,” says Floyd E. Romesberg, a chemist at Scripps Research Institute, La Jolla, Calif., who also studies directed evolution. Having the ability to control the selection pressure “is a significant step toward making this a practical method for the evolution of anything you can couple to production of the pIII protein.”



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