DNA Assembler

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 et al., 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.

Recent advances in natural product discovery

“…Recently, in the RecET direct cloning method, one strong promoter was added in front of the whole cluster to stimulate the production of natural products [36]. With the development of powerful DNA assembly methods such as DNA assembler [37], more comprehensive reconstruction of clusters has been achieved in a single-step manner, which results in the creation of novel compounds by combinatorial biosynthesis [38] and activation of a silent cluster for known compounds [39]. A similar strategy has been applied to discover novel polycyclic tetramate macrolactams (PTMs) [40].…”

DNA assembly techniques for next-generation combinatorial biosynthesis of natural products

“…An example is the application of the DNA Assembler method for combinatorial biosynthesis of the nitroaryl polyketide aureothin ( 1 ; Fig. 3) and its derivatives [39,40]. Modification of the domains of a multimodular PKS (either by mutation or replacement) is a well-established approach in combinatorial biosynthesis for the generation of derivatives of a target polyketide.…”

AFEAP cloning: a precise and efficient method for large DNA sequence assembly

“…The increasingly high demand for assembling large DNA into functional devices requires the methods that allow scarless, sequence independent, multi-fragment assembly of large constructs at high efficiency and high fidelity [ 6 , 7 ]. In the past decade, many novel DNA assembly methods, such as: Gibson Assembly (GA) [ 8 ], Golden Gate assembly [ 9 ], uracil-specific excision reagent cloning (USER) [ 10 ], ligase cycling reaction (LCR) [ 11 ], DNA assembler [ 12 ], twin-primer assembly (TPA) [ 6 ], sequence and ligation-independent cloning (SLIC) [ 13 ], seamless ligation cloning extract (SliCE) [ 14 ], enzyme-free cloning (EFC) [ 15 ], polymerase incomplete primer extension (PIPE) [ 16 ], in Vivo assembly (IVA cloning) [ 17 ], DNA assembly with thermostable exonuclease and ligase (DATEL) [ 7 ], and overlap extension PCR and recombination (OEPR Cloning) [ 18 ], have been designed and developed (Additional file 1 : Table S1), which opened doors to a wide variety of applications. These methods differ in both mechanism and scale, providing the effective means to cope with different needs [ 2 , 4 ].…”