Automated Construction of a Yeast-Based Multigene Library via Homologous Recombination in a Biofoundry Workflow

Efficiently building metabolic pathways via multigene assembly has long been constrained by the limitations of traditional cloning techniques, necessitating a breakthrough in gene assembly methods. Notably, various in vitro gene assembly methods have been developed to simplify the construction of an expression-tunable library. However, in vitro gene assembly requires a tedious multistep construction process, making it time-consuming and labor-intensive. Therefore, in this study, we developed an automated one-step multigene assembly method for constructing an expression-tunable library based on in vivo homologous recombination. We optimized the shuttle vector for in vivo homologous recombination to improve the assembly efficiency. We also scaled down the whole assembly method for a high-throughput gene assembly. Finally, the developed method demonstrated the construction of the expression-tunable multigene library in the biofoundry. Therefore, this study offers a versatile strategy for parallel and high-throughput genetic engineering in synthetic biology.