Bioscience Evidence 2024, Vol.14, No.3, 131-142 http://bioscipublisher.com/index.php/be 133 enzyme properties, such as in the case of β-lactamase, where DNA shuffling led to a 32,000-fold increase in antibiotic resistance. DNA shuffling is particularly effective when combined with iterative cycles of selection and backcrossing to eliminate non-essential mutations. 3.2 Site-directed mutagenesis Site-directed mutagenesis allows for the introduction of specific mutations at predetermined sites within a gene. This method is more targeted compared to random mutagenesis and can be used to explore the effects of individual amino acid changes on enzyme function. Site-saturation mutagenesis is a variant where all possible amino acid substitutions are introduced at specific positions. This approach has been shown to be more efficient than DNA shuffling for certain applications. For example, a single round of site-saturation mutagenesis and screening identified β-fucosidases with significantly higher activity and specificity compared to variants obtained through multiple rounds of DNA shuffling (Parikh and Matsumura, 2005; Jakočiūnas et al., 2018). This method is particularly useful for fine-tuning enzyme properties and exploring the functional landscape of specific residues. 3.3 High-throughput screening methods High-throughput screening (HTS) is essential for identifying improved enzyme variants from large mutagenesis libraries. These methods enable the rapid and efficient evaluation of thousands to millions of variants. Fluorescence-based assays are commonly used in HTS due to their sensitivity and ease of automation. These assays can detect changes in enzyme activity by measuring the fluorescence intensity of substrates or products. For example, fluorescence-based screening was used to identify mCherry fluorescent protein mutants with altered colors and intensities, demonstrating the utility of this approach in directed evolution (Kim et al., 2016; Yang et al., 2017). Microfluidic devices offer another powerful HTS platform, allowing for the miniaturization and parallelization of screening assays. These devices can handle small volumes and enable the rapid processing of large libraries. Microfluidic HTS has been successfully applied to evolve enzymes with enhanced catalytic properties, providing a scalable and efficient method for directed evolution (Figure 1) (Zeymer and Hilvert, 2018). By combining these methods, researchers can effectively explore the vast sequence space of enzymes, leading to the discovery of variants with improved catalytic efficiency and novel functionalities. 4 Synthetic Biology Tools for Directed Evolution 4.1 CRISPR/Cas systems CRISPR/Cas systems have revolutionized genome engineering by providing a highly precise and programmable method for gene editing. These systems utilize RNA-guided nucleases, such as Cas9, to introduce double-strand breaks at specific genomic locations, which can then be repaired to create mutations or insertions. This technology has been widely adopted in various fields, including plant biology, where it has been used to incorporate desirable traits into crops (Kumlehn et al., 2018). Additionally, CRISPR/Cas systems have been employed to enhance the catalytic efficiency of enzymes through directed evolution, as demonstrated by the development of catalytically enhanced Cas9 variants (Hand et al., 2020). The versatility of CRISPR/Cas systems extends to multiplexed applications, allowing simultaneous editing of multiple genomic sites, which is particularly useful for complex metabolic engineering tasks (Figure 2) (McCarty et al., 2020). 4.2 Recombinant DNA technology Recombinant DNA technology forms the backbone of synthetic biology by enabling the manipulation and recombination of genetic material. This technology allows for the construction of mutant libraries and the expression of recombinant proteins, which are essential for directed evolution experiments. For instance, the integration of multigene biosynthetic pathways into the genome of Pichia pastoris has been achieved using recombinant DNA techniques combined with CRISPR/Cas9, facilitating the production of valuable compounds (Gao et al., 2022). The ability to create and screen large libraries of genetic variants accelerates the identification of enzyme variants with improved catalytic properties (Zeymer and Hilvert, 2018; Shanmugam et al., 2020).
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