Methods in Enzymology, Volume 644, the latest release in this ongoing serial, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Chapters in this new release include Site-directed recombination (SDR) in vivo: a fast and reliable tool to unveil beneficial epistasis, Creation and application of amine oxidase with expanded substrate specificities from porcine kidney D-amino acid oxidase, Methods to assess correlation networks for engineering transketolase, Exploration of Enzyme Diversity by Integrating Bioinformatics with Microfluidics, Engineering lytic polysaccharide monooxygenases (LPMOs), Emulsion-based directed evolution of enzymes in yeast, and much more.