title: DNA assembler: a synthetic biology tool for characterizing and engineering natural product gene clusters.
abstract: The majority of existing antibacterial and anticancer drugs are natural products or their derivatives. However, the characterization and engineering of these compounds are often hampered by limited ability to manipulate the corresponding biosynthetic pathways. Recently, we developed a genomics-driven, synthetic biology-based method, DNA assembler, for discovery, characterization, and engineering of natural product biosynthetic pathways (Shao, Luo, & Zhao, 2011). By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in individual hosts in a single-step manner. In this chapter, we describe the general guidelines for construct design. By using two distinct biosynthetic pathways, we demonstrate that DNA assembler can perform multiple tasks, including heterologous expression, introduction of single or multiple point mutations, scar-less gene deletion, generation of product derivatives, and creation of artificial gene clusters. As such, this method offers unprecedented flexibility and versatility in pathway manipulations.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Zengyi
authors.initials: Z
authors.lastname: Shao
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002867:Chromones / Q000032:analysis / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D023281:Genomics / Q000379:methods*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D011753:Pyrones / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D015533:Transcriptional Activation; D014170:Transformation, Genetic
chemical_list: D001688:Biological Products; D002867:Chromones; D004269:DNA, Bacterial; D011753:Pyrones; C508401:spectinabilin; C012198:aureothin