Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters

Abstract

Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.

Extremophilic Fungi from Marine Environments: Underexplored Sources of Antitumor, Anti-Infective and Other Biologically Active Agents

“…This fermentation method takes advantage of symbiotic or competitive growth conditions to trigger secondary metabolite production via upregulating silent genes. Regarding screening and culture-independent approaches, readers should consult the recent paper by Nie et al [152], the reviews by Lage et al [153] and Wang et al [154], to see how these research areas are also providing investigators with tools to access bioactive fungal metabolites.…”

Recent Advances in the Heterologous Expression of Biosynthetic Gene Clusters for Marine Natural Products

“…The Gibson assembly [ 121 ] is another in vitro method for joining multiple DNA fragments with the help of 5′ exonuclease ( Figure 2 d). Wang [ 122 ] systematically and comprehensively reviewed the latest BGC cloning methods.…”

Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins

“…The robust modular and combinatorial advantages of GG have led to its application in synthetic biology systems with many recombinant parts, such as polycistronic vectors or engineered multigene metabolic circuits. Over the past decade, GG toolkits have become the dominant recombinant technology for building complex genetic constructs in microbes with increasing applications in plants and animals. − …”