Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR

DNA engineering is the fundamental motive driving the rapid development of modern biotechnology. Here, we present a versatile evolution method termed "rapidly efficient combinatorial oligonucleotides for directed evolution" (RECODE) for rapidly introducing multiple combinatorial mutations to the target DNA by combined action of a thermostable high-fidelity DNA polymerase and a thermostable DNA Ligase in one reaction system. By applying this method, we rapidly constructed a variant library of the rpoS promoters (with activity of 8–460%), generated a novel heparinase from the highly specific leech hyaluronidase (with more than 30 mutant residues) and optimized the heme biosynthetic pathway by combinatorial evolution of regulatory elements and pathway enzymes (2500 ± 120 mg L–1 with 20-fold increase). The simple RECODE method enabled researchers the unparalleled ability to efficiently create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.