Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces

Jian Wang, Ke Wang, Zhe Deng, Zhiyu Zhong, Guo Sun, Qing Mei, Fuling Zhou, Zixin Deng and Yuhui Sun ()
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Jian Wang: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Ke Wang: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Zhe Deng: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Zhiyu Zhong: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Guo Sun: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Qing Mei: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Fuling Zhou: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Zixin Deng: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University
Yuhui Sun: Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: Abstract Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.

Date: 2024
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DOI: 10.1038/s41467-024-49987-3

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