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

Abstract

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.

Microbial genome mining for accelerated natural products discovery: is a renaissance in the making?

“…Heterologous expression can often aid in the production of adequate levels of compound for evaluation, particularly when expression hosts are derived from industrial production strains or highly engineered laboratory strains [9, 11, 34, 40]. Synthetic biology approaches are also being developed which focus on refactoring secondary metabolic gene clusters, either in producing hosts via genetic recombination, or in ‘clean’ heterologous hosts via gene synthesis [22, 62]. Heterologous expression approaches have many advantages in that they are not limited to gene clusters derived from cultivatable microbes, that the products of heterologous expression can be identified by comparatively straightforward differential metabolomic analysis of the clean and transformed host, and that heterologous systems, once established, can be readily genetically manipulated to diversify the encoded natural products.…”

“…This special issue addresses both major categories of approaches. The work of Zhu et al [76], Ochi et al [61], and Yoon and Nodwell [72] focus specifically on creative new methods to activate secondary metabolism in actinomycetes and the work of Gomez-Escribo and Bibb [34], Ikeda et al [40] and Cobb et al [22] discuss cutting edge approaches to heterologous expression.…”

Recent advances in natural product discovery

“…However, the uncultivable, intractable majority of microorganisms remains a significant obstacle to this approach. With the development of powerful DNA assembly techniques [53], the future will likely see an increase not only in the heterologous expression of full gene clusters, but also the complete redesign and refactoring of these clusters in well-studied, highly tractable hosts tailor-made for heterologous expression.…”

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

“…Briefly, target natural product biosynthetic gene clusters are identified from sequenced genomes using powerful computational tools such as AntiSMASH 5, 6 . Various DNA assembly tools 7, 8 , along with engineered heterologous hosts 3 are then used for reconstruction and expression of those large biosynthetic gene clusters. To identify the target natural products, the resulting metabolite profiles are evaluated and characterized by advanced powerful metabolomics and detection techniques 9, 10 .…”