Recent advances in high-throughput metabolic engineering: Generation of oligonucleotide-mediated genetic libraries

The preparation of genetic libraries is an essential step to evolve microorganisms and study genotype-phenotype relationships by high-throughput screening/selection. As the large-scale synthesis of oligonucleotides becomes easy, cheap, and high-throughput, numerous novel strategies have been developed in recent years to construct high-quality oligo-mediated libraries, leveraging state-of-art molecular biology tools for genome editing and gene regulation. This review presents an overview of recent advances in creating and characterizing in vitro and in vivo genetic libraries, based on CRISPR/Cas, regulatory RNAs, and recombineering, primarily for Escherichia coli and Saccharomyces cerevisiae. These libraries' applications in high-throughput metabolic engineering, strain evolution and protein engineering are also discussed.