Directed Evolution of Replication-Competent Double-Stranded DNA Bacteriophage toward New Host Specificity

Abstract

In the fight against antimicrobial resistance, bacteriophages are a promising alternative to antibiotics. However, due to their narrow spectra, phage therapy requires the careful matching between the host and bacteriophage to be effective. Despite our best efforts, nature remains as the only source of novel phage specificity. Directed evolution can potentially open an avenue for engineering phage specificity and improving qualities of phages that are not strongly selected for in their natural environments but are important for therapeutic applications. In this work, we present a strategy that generates large libraries of replication-competent phage variants directly from synthetic DNA fragments, with no restriction on their host specificity. Using the T7 bacteriophage as a proof-of-concept, we created a large library of tail fiber mutants with at least 107 unique variants. From this library, we identified mutants that have broadened specificity as evidenced by their novel lytic activity against Yersinia enterocolitica, a strain that the wild-type T7 was unable to lyse. Using the same concept, mutants with improved lytic efficiency and characteristics, such as lytic condition tolerance and resistance suppression, were also identified. However, the observed limitations in altering host specificity by tail fiber mutagenesis suggest that other bottlenecks could be of equal or even greater importance.

PHEIGES, all-cell-free phage synthesis and selection from engineered genomes

“…PHEIGES provides a rapid, technically accessible, and low-cost method for phage engineering. Its DNA assembly efficiency (∼10 10 PFU/ml) offers many advantages compared to the current in vivo methods 15 15 18 19 and other cell-free phage engineering methods 21 40 63 . The yeast cloning approach to phage genome engineering takes on the order of one week to achieve 64 65 .…”

“…Taking into account the genome synthesis, the GGA method takes more than one week. The GGA method is also not compatible with TXTL, consequently the assembly reaction must be cleaned up first, a step that considerably reduces the yields 63 , which is also the major limitation to the Gibson assembly method for phage engineering (< 10 5 PFU/ml) 40 . The PHEIGES workflow presented in this work eliminates all these steps.…”

Approaches for bacteriophage genome engineering

“…Cell-free approaches also shorten the design-build-test cycle, as they do not rely on growth and manipulation of a bacterial strain. Recently, T7 phage particles were generated in TXTL from a Gibson-assembled genome containing a modified tail fiber gene [81], aiming at expanding the host range (Box 1). In vitro phage tail engineering is currently limited by retaining the relation between genotype and phenotype during rebooting to enable tracing of a successful phage particle within a large library…”

Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions

“…The in vitro synthesis approach provides a highly efficient and precise method for modifying specific gene sequences within bacteriophage genomes. Notable examples of this approach include the modular assembly and replacement of RBPs in bacteriophage genomes using yeast cell systems ( Ando et al, 2015 ), as well as the construction of mutated bacteriophage gene libraries through golden gate assembly ( Liang et al, 2022 ). One of the major advantages of in vitro synthesis is its superior efficiency compared to intracellular recombination within host cells.…”