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发帖数: 1187 | 1 A system for the continuous directed evolution of
biomolecules
Kevin M. Esvelt1, Jacob C. Carlson2 & David R. Liu2,3
Laboratory evolution has generated many biomolecules with
desired properties, but a single round of mutation, gene expression,
screening or selection, and replication typically requires days or
longer with frequent human intervention1. Because evolutionary
success is dependent on the total number of rounds performed2, a
means of performing laboratory evolution continuously and
rapidly could dramatically enhance its effectiveness3. Although
researchers have accelerated individual steps in the evolutionary
cycle4–9, the only previous example of continuous directed evolution
was the landmark study of Wright and Joyce10, who continuously
evolvedRNA ligase ribozymes with anin vitro replication cycle that
unfortunately cannot be easily adapted to other biomolecules. Here
we describe a systemthat enables the continuous directed evolution
of gene-encoded molecules that can be linked to protein production
in Escherichia coli. During phage-assisted continuous evolution
(PACE), evolving genes are transferred from host cell to host cell
through a modified bacteriophage life cycle in a manner that is
dependent on the activity of interest. Dozens of rounds of evolution
can occur in a single day of PACE without human intervention.
Using PACE, we evolved T7 RNA polymerase (RNAP) variants that
recognize a distinct promoter, initiate transcripts with ATP instead
of GTP, and initiate transcripts with CTP. In one example, PACE
executed 200 rounds of protein evolution over the course of 8 days.
Starting from undetectable activity levels in two of these cases,
enzymes with each of the three target activities emerged in less than
1 week of PACE. In all three cases, PACE-evolved polymerase
activities exceeded or were comparable to that of the wild-type T7
RNAP on its wild-type promoter, representing improvements of up
to several hundred-fold. By greatly accelerating laboratory evolution,
PACE may provide solutions to otherwise intractable directed
evolution problems and address novel questions about molecular
evolution. |
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