title: Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR.
abstract: 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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jin
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Kang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Junli
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Linpei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D019020:Directed Molecular Evolution; D005818:Genetic Engineering; D019763:Heparin Lyase / Q000235:genetics* / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000235:genetics* / Q000378:metabolism; D007865:Leeches / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity
chemical_list: D004259:DNA-Directed DNA Polymerase; D006821:Hyaluronoglucosaminidase; D019763:Heparin Lyase; D011088:DNA Ligases
keywords: combinatorial mutagenesis; directed evolution; pathway optimization; single-stranded DNA oligonucleotides; synthetic biology