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CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.
He, Fuqiang; Liu, Xinpeng; Tang, Min; Wang, Haiyi; Wu, Yun; Liang, Shufang.
Afiliación
  • He F; Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, P.R. China.
  • Liu X; Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, P.R. China.
  • Tang M; Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, P.R. China.
  • Wang H; Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, P.R. China.
  • Wu Y; Department of Cell Biology, College of Life Science, Sichuan Normal University, Chengdu, Sichuan, 610101, P.R. China.
  • Liang S; Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, P.R. China.
Nucleic Acids Res ; 2024 Sep 13.
Article en En | MEDLINE | ID: mdl-39271125
ABSTRACT
The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Nucleic Acids Res Año: 2024 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Nucleic Acids Res Año: 2024 Tipo del documento: Article Pais de publicación: Reino Unido