title: Construction and engineering of large biochemical pathways via DNA assembler.
abstract: DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Zengyi
authors.initials: Z
authors.lastname: Shao
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics*; D002867:Chromones / Q000378:metabolism; D014644:Genetic Variation; D059765:Homologous Recombination; D060847:Metabolic Engineering / Q000379:methods*; D005810:Multigene Family; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D014170:Transformation, Genetic; D024341:Xanthophylls / Q000096:biosynthesis; D065146:Zeaxanthins
chemical_list: D002867:Chromones; D024341:Xanthophylls; D065146:Zeaxanthins; C012198:aureothin