A system for the continuous directed evolution of biomolecules

Abstract

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 intervention. Because evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that 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.

Figure 1

Overview of the PACE system. PACE in a single lagoon. Host cells continuously flow through a lagoon, where they are infected with selection phage (SP) encoding library members. Functional library members induce production of pIII from the accessory plasmid (AP) and release progeny capable of infecting new host cells, while non-functional library members do not. Increased mutagenesis is triggered through induction of the mutagenesis plasmid (MP). Host cells flow out of the lagoon on average faster than they can replicate, confining the accumulation of mutations to replicating phage.

Figure 2

Linkage of three protein activities to pIII production and phage infectivity using three distinct APs. E. coli cells containing APs encoding conditionally expressed gene III (left) and selection phage were combined with recipient cells. Phage production resulted in colonies with antibiotic resistances conferred by the phage and the recipient cells (right). See Methods for details. (a) RNA polymerase activity leads to gene III expression and infection comparable to wild-type phage, while SP lacking T7 RNAP do not infect. (b) Protein-protein interaction between a Gal11p domain tethered to a Zif268 DNA-binding domain and an LGF2a domain fused to RNA polymerase leads to gene III expression and infection. (c) Recombinase-catalysed gene inversion induces gene III expression and infection.

Figure 3

Continuous evolution of T7 RNAP variants that recognize the T3 promoter. (a) PACE schedule. (b) Activity in cells of T7 RNAP variants isolated from lagoon 1 at 48, 108, and 192 hours on the T7 and T3 promoters. Transcriptional activity was measured spectrophotometrically by subcloning the protein-encoding regions of the T7 RNAP genes into a construct in which the T7 or T3 promoter drives lacZ expression. (c) Activity in cells of T7 RNAP variants isolated from lagoon 2. Error bars in (b) and (c) represent the standard deviation of at least three independent assays. (d) Mutations identified in T7 RNAP clones from lagoons 1 and 2 are shown in red and blue, respectively. Underlined mutations were predominant in that lagoon based on whole-pool sequencing of lagoon aliquots. Mutations highlighted in yellow independently evolved to predominance in both lagoons.

Figure 4

Continuous evolution of T7 RNAP variants that initiate transcription with A. (a) PACE schedule. (b) Activity in cells of T7 RNAP variants on the T7 and iA₆ promoters isolated after 36 hours of PACE. Assays were performed as described in Fig. 3b. Error bars represent the standard deviation of at least three independent assays.