Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination

Zhang, Lin; Zhao, Guoping; Ding, Xiaoming

doi:10.1038/srep00141

Cited by 59 publications

(50 citation statements)

References 25 publications

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“…By synthetically generating four additional orthogonal att recombination sequences, Gateway has also recently been expanded to enable the cloning of multiple parts simultaneously 41 . Similar non-commercial systems have also been developed that use alternative phage integrases, including phiBT1 and phiC31 42,43 . For all these methods, reactions at recombined att sites can be reversed by additionally providing either an excisionase (a bacteriophage excision protein) or a recombination directionality factor (RDF), which is an accessory protein that in combination with the integrase reverses the reaction and leads to excision rather than integration.…”

Section: Site-specific Recombinationmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

247

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Section: Site-specific Recombinationmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

247

186

View full text Add to dashboard Cite

show abstract

“…A series of mutated attB sites are placed upstream of each module, and mutated attP sites are located downstream. After incubation of all the DNA modules with the φBT1 integrase, tandem assembled products are produced 23 . This approach can efficiently and accurately join multiple DNA molecules in a defined order in vitro .…”

Section: Pathway Reconstruction In Heterologous Hostsmentioning

confidence: 99%

New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

2016

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Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering.

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“…After several cycles, the nicked target construct is formed, which can be sealed in vivo [36]. Site-specific recombination-based tandem assembly (SSRTA), on the other hand, employs the Streptomyces phage φBT1 integrase to splice neighboring fragments in vitro [46] (Fig. 2).…”

Section: Synthetic Biology Techniques For Dna Assemblymentioning

confidence: 99%

“…One example of an in vitro recombination-based assembly technique applied to construct a natural product pathway was provided by Zhang and coworkers [46], who utilized SSRTA to assemble the complete 56 kb epothilone gene cluster from Sorangium cellulosum So0157-2. They performed the assembly in two steps, first assembling the large PKS epoD from four fragments (plus a receiving vector), and then the full cluster from six fragments (plus a receiving vector).…”

Section: Current and Future Applications In Combinatorial Biosynthesismentioning

confidence: 99%

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

Cobb

Ning

Zhao

2014

Journal of Industrial Microbiology and Biotechnology

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Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently-developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis.

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Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination

Cited by 59 publications

References 25 publications

Bricks and blueprints: methods and standards for DNA assembly

Bricks and blueprints: methods and standards for DNA assembly

New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters

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

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