TY - CHAP
T1 - DNA assembler
T2 - A synthetic biology tool for characterizing and engineering natural product gene clusters
AU - Shao, Zengyi
AU - Zhao, Huimin
N1 - Funding Information:
This work was supported by the National Academies Keck Futures Initiative on Synthetic Biology and the National Institutes of Health (GM077596).
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - Aureothin
KW - Genetic manipulation of gene clusters
KW - Heterologous expression
KW - Natural product biosynthesis
KW - Spectinabilin
KW - Synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=84867645703&partnerID=8YFLogxK
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U2 - 10.1016/B978-0-12-404634-4.00010-3
DO - 10.1016/B978-0-12-404634-4.00010-3
M3 - Chapter
C2 - 23084940
AN - SCOPUS:84867645703
SN - 9780124046344
T3 - Methods in Enzymology
SP - 203
EP - 224
BT - Natural Product Biosynthesis by Microorganisms and Plants, Part C
PB - Academic Press Inc.
ER -