A system for the continuous directed evolution of biomolecules
Kevin M. Esvelt
, Jacob C. Carlson
& David R. Liu
Laboratory evolution has generated many biomolecules withdesiredproperties,butasingleroundofmutation,geneexpression,screening or selection, and replication typically requires days or longer with frequent human intervention
. Because evolutionary success is dependent on the total number of rounds performed
, a means of performing laboratory evolution continuously andrapidly could dramatically enhance its effectiveness
. Althoughresearchers have accelerated individual steps in the evolutionary cycle
,theonlypreviousexampleofcontinuousdirectedevolutionwas the landmark study of Wright and Joyce
, who continuously evolvedRNAligaseribozymeswithan
. During phage-assisted continuous evolution(PACE), evolving genes are transferred from host cell to host cellthrough a modified bacteriophage life cycle in a manner that isdependentontheactivityofinterest.Dozensofroundsofevolutioncan occur in a single day of PACE without human intervention.UsingPACE,weevolvedT7RNApolymerase(RNAP)variantsthatrecognizeadistinctpromoter,initiatetranscriptswithATPinsteadof GTP, and initiate transcripts with CTP. In one example, PACEexecuted 200 rounds of protein evolution over the course of 8days.Starting from undetectable activity levels in two of these cases,enzymeswitheachofthethreetargetactivitiesemergedinlessthan1week of PACE. In all three cases, PACE-evolved polymeraseactivities exceeded or were comparable to that of the wild-type T7RNAPonitswild-typepromoter,representingimprovementsofupto several hundred-fold. By greatly accelerating laboratory evolu-tion,PACEmayprovidesolutionstootherwiseintractabledirectedevolution problems and address novel questions about molecular evolution.
We devised a system that exploits the continuous culture and selec-tionoftheM13filamentousbacteriophage
) to enable the continuous directed evolution of proteins ornucleic acids. In PACE,
host cells continuously flow through afixed-volume vessel (the ‘lagoon’) containing a replicating populationof phage DNA vectors (‘selection phage’) encoding the gene(s) of interest (Supplementary Fig. 1).Theaverageresidencetimeofhostcellsinthelagoonislessthanthetime required for
replication. As a result, mutations accumulateonlyintheevolvingselectionphagepopulation,theonlyDNAthatcanreplicate faster than the rate of lagoon dilution. The mutation of hostcells in the lagoon should therefore have minimal impact on the out-come of the selection over many rounds of phage replication, andmutagenesis conditions are not limited to those that preserve
viability.PACE achieves continuous selection by linking the desired activity to the productionof infectious progeny phage containing the evolving gene(s).PhageinfectionrequiresproteinIII(pIII;encodedbygeneIII),whichmediatesFpilusbindingandhostcellentry
.PhagelackingpIIIare approximately 10
-fold less infectious than wild-type phage
.Crucially, the production of infectious phage scales with increasing levels of pIII over concentrations spanning two ordersof magnitude
.TocouplepIIIproductiontotheactivityofinterest,wedeletedgeneIII from the phage vector and inserted it into an ‘accessory plasmid’present in the
host cells (see Supplementary Fig. 2 for plasmidmaps). The production of pIII from the accessory plasmid is depend-enton the activity of the evolving gene(s) on the selection phage. Only phagevectorsabletoinducesufficientpIIIproductionfromtheaccess-ory plasmid will propagate and persist in the lagoon (Fig. 1). BecausepIII expression level determines the rate of infectious phage produc-tion
, phage encoding genes that result in a higher level of pIII pro-duction will infect more host cells than phage encoding less activegenes.Owing to the speed of the phage life cycle (progeny phage produc-tionbeginsapproximately10minafterinfection)
,PACEcanmediatemany generations of selective phage replication in a single day. Weobserved activity-dependent phage vectors that tolerate lagoon flow rates up to 3.2 volumes per hour (Supplementary Fig. 3), correspond-ingtoanaverageof38phagegenerationsper24h(seetheSupplemen-tary Information for an analysis). More conservative flow rates of 2.0–2.5 volumes per hour allow 24–30 generations per day and reducethe risk of complete phage loss (washout) during selections. Multiplelagoons can evolve genes in parallel, with each 100ml lagoon contain-ing approximately 5
host cells selectively replicating activephage variants. Importantly, PACE requires no intervention during evolution and obviates the need to create DNA libraries, transformcells, extract genes or perform DNA cloning steps during each round.Inprinciple, PACEiscapableofevolvinganygenethatcanbelinkedto pIIIproductionin
. Because a widevarietyoffunctions includ-ing DNA binding, RNA binding, protein binding, bond-forming cata-lysis and a variety of enzyme activities have been linked to the expres-sion of a reporter protein
, PACE can be applied to the evolution of manydifferentactivitiesofinterest.Asexamples,wesuccessfullylinkedprotein–protein binding, recombinase activity and RNAP activity tophage infectivity in discrete infection assays by creating variants of the accessory plasmid that associate each of these activities with pIIIproduction (Fig. 2).PACE applies optimal evolutionary pressure when pIII levels areabove the minimal threshold required to prevent phage washout, butbelow the amount needed to maximize infectious phage production.Thiswindowcanbeshiftedbyvaryingthecopynumberoftheaccessory plasmid, or by altering the ribosome-binding site (RBS) sequence of gene III to modulate the efficiency with which gene III is expressed(Supplementary Fig. 4).We constructed an arabinose-inducible mutagenesis plasmid thatelevates the error rate during DNA replication in the lagoon by sup-pressingproofreading
. Full induction increased the observedmutagenesis rate by approximately 100-fold, inducing all possibletransitions and transversions (Supplementary Fig. 5). This enhanced
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge,Massachusetts 02138, USA.
Howard Hughes Medical Institute, Cambridge, Massachusetts 02138, USA.
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