Refactoring the Silent Spectinabilin Gene Cluster Using a Plug-and-Play Scaffold

Abstract

Natural products (secondary metabolites) are a rich source of compounds with important biological activities. Eliciting pathway expression is always challenging but extremely important in natural product discovery because individual pathway is tightly controlled through unique regulation mechanism and hence often remains silent in the routine culturing conditions. To overcome the drawback of the traditional approaches that lack general applicability, we developed a simple synthetic biology approach that decouples pathway expression from complex native regulations. Briefly, the entire silent biosynthetic pathway is refactored using a plug-and-play scaffold and a set of heterologous promoters that are functional in a heterologous host under the target culturing condition. Using this strategy, we successfully awakened the silent spectinabilin pathway from Streptomyces orinoci. This strategy bypasses the traditional laborious processes to elicit pathway expression and represents a new platform for discovering novel natural products.

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

“…Overexpression of key enzymes [46] and regulatory activators [47] has been reported to be effective strategies to improve antibiotics production. In this study, the sfp gene was overexpressed in B-9987 under the control of the strong constitutive promoter gapDHp, whose counterpart in Streptomyces has recently been proved to be much stronger than the commonly-used promoter ermE*p [48]. Thus, macrolactins A production was increased by 1.6-fold compared with that of the wild type strain (Figure 4), which potentially enhanced the biocontrol ability of B-9987.…”

The re-emergence of natural products for drug discovery in the genomics era

“…Indeed, metagenomic approaches have now become essential tools for elucidating, defining, and controlling biosynthetic pathways 131 . A key goal of these approaches is to develop an efficient plug and play heterologous gene expression platform that will facilitate the production of particular target secondary metabolites 149,150 or a plethora of new natural products 111, 151-154 .…”

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…Impressively, a brand new functional bacterial genome and eukaryotic chromosome have been successfully assembled using rational design and chemical synthesis. 33,34 Through random assembly of a set of constitutive promoters, a silent spectinabilin pathway from Streptomyces orinoci and a cryptic polycyclic tetramate macrolactams biosynthetic gene cluster from Streptomyces griseus were respectively discovered and characterized, 35,36 which confirmed the powerful ability of DNA-assembly methods in the discovery of novel natural products. More recently, using both combinatorial transcriptional engineering and directed evolution strategies, a library of promoters with varying strengths were assembled with the xylose-utilizing pathway or the cellobiose-utilizing pathway functional structural genes.…”