Continuous In Vitro Directed Evolution of Macromolecules

Evolutionary processes, and specifically selection-based mechanisms, have long served to inspire in vitro methods for generating proteins and nucleic acids that mediate functions of interest.  Examples going back two decades include the development of phage display methods (Scott and Smith, 1990) and methods based on selection of RNA molecules (Ellington and Szostak, 1990; Tuerk and Gold, 1990) from libraries of enormous  structural diversity (as many as10¹⁰ distinct molecular structures).  A recent review (Dreier and Plückthun, 2011) provides a sense of what can be accomplished with one these methods, ribosome display, in terms of generating macromolecules, such as antibody fragments, with desired functional properties (e.g., high-affinity binding to a target molecule).

However, these methods typically involve repeated and somewhat tedious interventions by the investigators to progress from one round of molecular variant generation and selection to the next.  The overall process can be quite time-consuming.  An ideal method would permit continuous directed evolution of proteins or nucleic acids with minimal input from investigators once the system was “put in motion.”
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The recent Nature paper (2011) by Esvelt et al., from the laboratory of David Liu, provides what may be the first widely-applicable method for continuous in vitro variant generation and functional selection, i.e. continuous directed evolution in vitro.  More than a decade ago, Wright and Joyce described (Science, 1997) a method for the continuous directed evolution of catalytic RNA molecules, but as Liu and colleagues point out, this method is not readily applied to protein molecules.  In contrast, Esvelt et al. state that their procedures can be used to select “gene-encoded molecules that can be linked to protein production in Escherichia coli.”  Since both proteins and nucleic acids can be selected using  their methods, evolution of a wider array of macromolecules mediating a wider array of functions is potentially a realistic prospect.
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Liu and colleagues call their system for continuous directed evolution of biomolecules “phage-assisted continuous evolution,” or PACE.  It involves the M13 filamentous phage replicating in E. coli cells that continuously flow through a fixed-volume chamber that is referred to as the ‘lagoon.’  The genes encoding the selected biomolecules are contained in the M13 phage particles.

PACE is designed to minimize the effects of mutations in the E. coli cells because the average time these cells reside in the lagoon is less than the average time required for E. coli replication.  Selection is continuous because the replication of the M13 phage is orchestrated so as to be directly dependent on the activity of the biomolecule of interest.  This arrangement is achieved by using the activity of interest to regulate production of a phage protein, protein III or pIII, crucial to the infection of host E. coli cells.  The phage genomes proper are missing the gene III, which encodes pIII, but an accessory plasmid in the host cells encodes pIII.  Fortuitously, the rate of production of infectious phage particles increases directly with the concentration of intracellular pIII over a range of roughly 100-fold.
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The rate of evolution is rapid, as the time interval between host cell infection and production of new phage particles can be as brief as 10 minutes.  Variations in the flow rate of cells through the lagoon can be used to vary the number of phage generations per day (which can potentially reach or slightly exceed 30-35), but increasing the flow rate too high can result in the loss of the phage, ending the evolutionary process.

The authors have also engineered what they refer to as a mutagenesis plasmid into the host cells.  This plasmid-mediated function is inducible with arabinose and leads to a decrease in a proofreading function that normally operates during DNA replication.  Maximal induction of the the plasmid can boost the mutation rate by about 100-fold.  Anyone interested in other technical considerations should consult the article directly.

Esvelt et al.used PACE to evolve bacteriophage T7 RNA polymerases with new DNA sequence (i.e., promoter) specificities and other characteristics.  In one instance, the activity of the selected T7 RNA polymerase exhibited an improvement of 89-fold on the ‘new’ promoter.  These experiments were completed in anywhere from one and one-half to eight days and required minimal input from the investigators. Evolutionary medicines generated by such in vivo methods are now conceivable, and it will be interesting to follow the progress in this developing field of research.

References

Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science. 1990 Jul 27;249(4967):386-90. PubMed PMID: 1696028.

Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818-22. PubMed PMID: 1697402.

Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment:

RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505-10. PubMed PMID: 2200121.

Dreier B, Plückthun A. Ribosome display: a technology for selecting and evolving proteins from large libraries. Methods Mol Biol. 2011;687:283-306. PubMed PMID: 20967617.

Wright MC, Joyce GF. Continuous in vitro evolution of catalytic function. Science. 1997 Apr 25;276(5312):614-7. PubMed PMID: 9110984.

Esvelt KM, Carlson JC, Liu DR. A system for the continuous directed evolution of biomolecules. Nature. 2011 Apr 28;472(7344):499-503. Epub 2011 Apr 10. PubMed PMID: 21478873; PubMed Central PMCID: PMC3084352.