Cell-free expression system: a promising platform for bacteriophage production and engineering

Abstract Cell-free expression is a technique used to synthesize proteins without utilising living cells. This technique relies mainly on the cellular machinery —ribosomes, enzymes, and other components — extracted from cells to produce proteins in vitro. Thus far, cell-free expression systems have been used for an array of biologically important purposes, such as studying protein functions and interactions, designing synthetic pathways, and producing novel proteins and enzymes. In this review article, we aim to provide bacteriophage (phage) researchers with an understanding of the cell-free expression process and the potential it holds to accelerate phage production and engineering for phage therapy and other applications. Throughout the review, we summarize the system’s main steps and components, both generally and particularly for the self-assembly and engineering of phages and discuss their potential optimization for better protein and phage production. Cell-free expression systems have the potential to serve as a platform for the biosynthetic production of personalized phage therapeutics. This is an area of in vitro biosynthesis that is becoming increasingly attractive, given the current high interest in phages and their promising potential role in the fight against antimicrobial resistant infections.