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, 2 (11), 662-9

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

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

Zengyi Shao et al. ACS Synth Biol.

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 an individual pathway is tightly controlled through a unique regulation mechanism and hence often remains silent under the routine culturing conditions. To overcome the drawbacks 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.

Figures

Figure 1
Figure 1
Promoter screening for pathway refactoring in Streptomycetes. a) Identification of strong constitutive promoters by real-time PCR analysis of the transcription of 23 housekeeping genes in S. griseus. Samples were taken at different time points. The y-axis scale represents the expression value relative to that of HrdB, a commonly used housekeeping sigma factor (–58), which was set to 1. The expressions of gapdh and rpsL were higher than the other genes at all the sampling points, with the 12-hour samples showing the highest level of significance (46- and 27-fold higher than that of hrdB, respectively). b) Evaluation of the activities of the heterologous promoters using xylE as a reporter. The entire intergenic region between the target gene and its upstream gene was cloned upstream of xylE. Here we did not experimentally determine the ribosomal binding site (RBS) for each promoter and assumed it is located 6–10 bp upstream of each start codon. However, we did find these intergenic regions are AG-rich for most promoters. Promoters actIp and ermE*p are the two commonly used promoters reported in literature (21, 22, 59). The two letters in parentheses represent the names of individual actinomycetes (Supporting Information Table S2).
Figure 2
Figure 2
(a) The spectinabilin gene cluster from S. orinoci. (b) Real-time PCR analysis of the nor gene transcription in S. lividans and S. orinoci.
Figure 3
Figure 3
a) The refactored spectinabilin pathway. b) LC-MS analysis of the extract from the S. lividans strain carrying the refactored nor pathway. The peak labeled by a star indicated the target product peak. c) Real-time PCR analysis of the nor gene transcription in the refactored pathway in S. lividans (For the purpose of direct comparison, the real-time PCR analysis of the nor gene transcription in S. orinoci illustrated in Figure 2b was incorporated).
Scheme 1
Scheme 1
Design of a plug-and-play scaffold for refactoring cryptic natural product biosynthetic pathways. The scaffold consists of promoter modules, gene modules and helper modules. The refactoring strategy is to select a single heterologous host, identify a set of strong promoters under a target culture condition, assemble individual biosynthetic genes with these promoters into a new gene cluster, and express the refactored gene cluster in the heterologous host under the target culture condition.

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