Presentation Information
[4GteX-13]DiPaSE: A one-pot DNA assembly method for accurate and efficient refactoring of high-GC, long BGCs
○Satoshi Yuzawa1 (1. Keio University (Japan))
Keywords:
Biosynthetic gene clusters,High-GC DNA assembly,Multiplex pathway editing,Natural product biosynthesis
[Purpose]
Actinobacterial genomes are characterized by high GC content and typically harbor 20–40 biosynthetic gene clusters (BGCs) per genome, encoding diverse natural products such as polyketides, peptides, and glycosides. While CRISPR/Cas-based genome editing has emerged as a promising approach to activate silent BGCs and engineer natural product biosynthesis, its efficiency markedly declines with increasing numbers of targeted loci, limiting multiplex pathway refactoring. This study aims to develop an efficient and scalable strategy for the synthesis and multiplex editing of long, high-GC BGCs to facilitate systematic functional analysis and rational engineering.
[Method]
We developed a one-pot DNA assembly platform, termed DiPaSE (Direct Pathway Synthesis and Editing), designed for accurate synthesis and refactoring of high-GC BGCs. DiPaSE employs standard PCR to generate ~5 kb DNA fragments, followed by cloning with type IIP restriction enzymes for sequence verification to eliminate PCR-derived mutations. Mutation-free fragments are excised and assembled in a single HiFi assembly reaction, assembling multiple DNA fragments with a total length of up to 60 kb. The workflow relies on conventional molecular biology techniques, commercially available reagents, and Escherichia coli as a host.
[Results]
Using DiPaSE, we successfully constructed plasmids harboring 10–55 kb GC-rich BGCs with high efficiency and accuracy; nearly all assembled constructs were mutation-free. In contrast, conventional one-step HiFi assembly without fragment validation showed reduced efficiency and introduced mutations. Importantly, multiplex insertions and deletions within the aureothin BGC did not compromise assembly efficiency. This approach enabled functional characterization of previously unannotated genes, demonstrated that multiple promoter insertions are required for effective pathway activation, and resulted in a marked increase in aureothin production.
[Consideration]
Our results indicate that the quality and purity of DNA fragments are critical determinants of successful assembly of long, high-GC BGCs. By decoupling pathway-scale DNA synthesis and multiplex editing from locus-specific genome manipulation, DiPaSE overcomes key limitations of CRISPR/Cas-based approaches, particularly the decline in efficiency with increasing target sites and the risk of off-target mutations. The ability to refactor BGCs without loss of efficiency also highlights the potential of DiPaSE for systematic pathway optimization.
[Conclusion]
DiPaSE provides a robust, accurate, and scalable one-pot platform for the synthesis and multiplex refactoring of long, high-GC BGCs. This method enables flexible modular reconstruction of BGCs and supports natural product discovery, pathway activation, functional characterization, and large-scale production of natural products and their analogs.
Actinobacterial genomes are characterized by high GC content and typically harbor 20–40 biosynthetic gene clusters (BGCs) per genome, encoding diverse natural products such as polyketides, peptides, and glycosides. While CRISPR/Cas-based genome editing has emerged as a promising approach to activate silent BGCs and engineer natural product biosynthesis, its efficiency markedly declines with increasing numbers of targeted loci, limiting multiplex pathway refactoring. This study aims to develop an efficient and scalable strategy for the synthesis and multiplex editing of long, high-GC BGCs to facilitate systematic functional analysis and rational engineering.
[Method]
We developed a one-pot DNA assembly platform, termed DiPaSE (Direct Pathway Synthesis and Editing), designed for accurate synthesis and refactoring of high-GC BGCs. DiPaSE employs standard PCR to generate ~5 kb DNA fragments, followed by cloning with type IIP restriction enzymes for sequence verification to eliminate PCR-derived mutations. Mutation-free fragments are excised and assembled in a single HiFi assembly reaction, assembling multiple DNA fragments with a total length of up to 60 kb. The workflow relies on conventional molecular biology techniques, commercially available reagents, and Escherichia coli as a host.
[Results]
Using DiPaSE, we successfully constructed plasmids harboring 10–55 kb GC-rich BGCs with high efficiency and accuracy; nearly all assembled constructs were mutation-free. In contrast, conventional one-step HiFi assembly without fragment validation showed reduced efficiency and introduced mutations. Importantly, multiplex insertions and deletions within the aureothin BGC did not compromise assembly efficiency. This approach enabled functional characterization of previously unannotated genes, demonstrated that multiple promoter insertions are required for effective pathway activation, and resulted in a marked increase in aureothin production.
[Consideration]
Our results indicate that the quality and purity of DNA fragments are critical determinants of successful assembly of long, high-GC BGCs. By decoupling pathway-scale DNA synthesis and multiplex editing from locus-specific genome manipulation, DiPaSE overcomes key limitations of CRISPR/Cas-based approaches, particularly the decline in efficiency with increasing target sites and the risk of off-target mutations. The ability to refactor BGCs without loss of efficiency also highlights the potential of DiPaSE for systematic pathway optimization.
[Conclusion]
DiPaSE provides a robust, accurate, and scalable one-pot platform for the synthesis and multiplex refactoring of long, high-GC BGCs. This method enables flexible modular reconstruction of BGCs and supports natural product discovery, pathway activation, functional characterization, and large-scale production of natural products and their analogs.
Comment
To browse or post comments, you must log in.Log in