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

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• 作者：Peng Jin ; Zhen Kang ; Junli Zhang ; Linpei Zhang ; Guocheng Du ; Jian Chen
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：March 18, 2016
• 年：2016
• 卷：5
• 期：3
• 页码：259-268
• 全文大小：753K
• ISSN：2161-5063

文摘

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.