CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters

Area of Science:

• Synthetic Biology
• Molecular Biology
• Natural Product Discovery

Background:

• Activating silent biosynthetic gene clusters (BGCs) is crucial for discovering new bioactive natural products.
• Efficient refactoring of BGCs presents a significant challenge in natural product research.

Purpose of the Study:

• To develop a simple, robust technique for multiplexed refactoring of natural product BGCs.
• To enhance the heterologous production of daptomycin through promoter engineering.

Main Methods:

• Developed the CRISPR/Cas9 and RecET-mediated Refactoring (CRISETR) technique.
• Combined clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with RecET for homologous recombination.
• Applied CRISETR for promoter engineering of the daptomycin BGC.

Main Results:

• CRISETR achieved simultaneous replacement of four promoter sites and marker-free replacement of a single promoter site.
• Promoter engineering of the daptomycin BGC led to a 20.4-fold increase in daptomycin yield.
• CRISETR demonstrated tolerance to repetitive sequences within complex BGCs.

Conclusions:

• CRISETR is an effective tool for refactoring natural product BGCs, including complex ones with repetitive sequences.
• This technique significantly accelerates the discovery of novel bioactive metabolites.
• CRISETR facilitates enhanced heterologous production of valuable compounds like daptomycin.