CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters

Abstract

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Recent advances in natural products exploitation in Streptomyces via synthetic biology

“…Subsequently, Mingzi Zhang et al. developed an efficient CRISPR/Cas9 knock‐in strategy for the efficient and precise insertions of the constitutive promoters upstream of the main biosynthetic operons or the pathway‐specific activators, and successfully triggered the production of various novel natural products in different Streptomyces species. Moreover, Yawei Zhao et al.…”

Breaking the silence: new strategies for discovering novel natural products

“…Traditional promoter knock-in strategies based on homologous double-crossover recombination are often time-consuming and labor-intensive especially when applied to streptomycetes [18,19]. Recently we developed a highly efficient clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system for multiplex genome editing in streptomycetes, either targeting at precise gene deletion [20] or promoter knock-in [21 •• ]. By applying this one-step strategy to five Streptomyces species, multiple silent BGCs belonging to different classes were activated, among which a novel pentangular polyketide from S. viridochromogenes was characterized [21 •• ].…”

“…Recently we developed a highly efficient clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system for multiplex genome editing in streptomycetes, either targeting at precise gene deletion [20] or promoter knock-in [21 •• ]. By applying this one-step strategy to five Streptomyces species, multiple silent BGCs belonging to different classes were activated, among which a novel pentangular polyketide from S. viridochromogenes was characterized [21 •• ]. Considering the common multi-operon architecture in BGCs, this strategy may be used to iteratively knock-in promoters and activate the silent full-length BGCs, while different combinations of activated operons (by promoter insertion) can also provide insights into the biosynthetic mechanism (Figure 2).…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…[51] The CRISPR-Cas9 was applied in an alternative fashion for the knock-in strategy in order to activate silent BGCs in multiple Streptomyces strains. [48] Replacing native promoters with the strong, constitutive promoter kasOp à using the CRISPR-Cas9 system led to the production of new compounds in S. roseosporus, Streptomyces venezuelae, and Streptomyces viridochromogenes. Furthermore, the compound isolated from S. viridochromogenes was found to be a novel pigmented PK produced from an otherwise silent BGC of the type II PKS.…”

“…The CRISPR-Cas9 technique can be applied for deletion of genes and gene clusters [45][46][47] in a multiplex manner, [45] reversible gene expression control, [47] and induction. [48] The optimized system relies on two components: the single guide RNA (sgRNA), which is a synthetic RNA consisting of a CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) complex, and is required for guiding Cas9 to modify the targeted genome sequence; and the endonuclease Cas9 or catalytically inactive dCas9, which upon interaction with the sgRNA, scans the genome for protospacer adjacent motif (PAM) sequences and the corresponding 20 bp recognition site. Upon recognition of the target DNA sequence, Cas9 binds to and cleaves the specific site in the genome whereas the inactive dCas9 will bind to the target DNA without sequence modifications.…”