2 Journal of Nucleic Acids Figure 1: Genotype (genetic information)-phenotype (protein synthesized from the gene and its function) linkage and screening techniques for directed evolution of proteins. Screening techniques are categorized as in vivo and in vitro approaches. In vivo Phage display Phage Cell surface display Living cell Protein Plasmid DNA Ribosome display Protein 5 mRNA Ribosome 3 mRNA display 5 mRNA Puromycin 3 In vitro Protein 5 Protein Microcompartment Double emulsion Liposome DNA (genotype) Protein (phenotype) Transcription and translation Substrate Product High-throughput screening method Fluorescence-activated cell sorter (FACS) Flow cell Laser − Nozzle + P1 P2 P3 P4 Library CIS display repA RepA protein 3 CIS ori Protein Microbead display Products are attached to DNA via the bead. Protein (phenotype) DNA (genotype) Bead Product Figure 2: The underlying concept for in vitro compartmentalization (IVC) using double emulsion or liposome (left) and microbead (right). In both cases, a ﬂuorescence-activated cell sorter (FACS) (center) is used for high throughput screening. exposure to a colorimetric detection reagent, and selectively sorted using a ﬂuorescence-activated cell sorter (FACS) [4, 5]. Phage display is another in vivo display technique that uses a phage for gene storage and protein display. In this technique, target proteins are fused with phage coat proteins (g8p or g3p) and displayed on a phage surface. These in vivo screening techniques have been applied to the directed evolution of proteins. However, these techniques are applicable to a limited number of proteins that are not toxic to growth of the host cell. Low transformation eﬃciency also limits genetic diversity (library size) by up to 108. To overcome these technical drawbacks in in vivo techniques, in vitro display was proposed as a new display technique [6, 7]. In this technique, protein variants are synthesized from the gene using an IVTT, and the gene (genotype) is physically or covalently tethered to the trans- lated protein (phenotype) via an adaptor or linker, such as ribosomes (ribosome display) [8], RepA (CIS display) [9], and puromycin (mRNA display) [10]. The proteins linked to the mutant gene are screened for the desired function. These in vitro display methods are suitable for improving protein equilibrium aﬃnity, oﬀ rate, stability, and folding [8]. However, these display techniques are not suitable for improving the catalytic activity of enzymes because they rely on binding aﬃnity between the displayed protein and an immobilized ligand for the screen [11]. In vitro compartmentalization (IVC) is a solution to direct screening for enzymatic reaction turnover entirely in vitro. The primary idea underlying IVC is that a DNA, an IVTT, and a ﬂuorogenic detection reagent are encapsulated in a cell-like compartment to form a genotype-phenotype linkage (Figure 2, left). Proteins are translated from a single gene using an IVTT in each compartment, and they yield a ﬂuorescent product that is screened directly for the catalytic activity of interest using an FACS [11]. We introduce herein the earlier studies on IVC-based directed evolution