A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host
Abstract Background Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. Results We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces...
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BMC
2025-05-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-025-02741-w |
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| author | Zhe-Chong Wang Hayden Stegall Takeshi Miyazawa Adrian T. Keatinge-Clay |
| author_facet | Zhe-Chong Wang Hayden Stegall Takeshi Miyazawa Adrian T. Keatinge-Clay |
| author_sort | Zhe-Chong Wang |
| collection | DOAJ |
| description | Abstract Background Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. Results We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin. Conclusion Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes. |
| format | Article |
| id | doaj-art-fd18d2fc9f0b4611a2eedc19606755f2 |
| institution | DOAJ |
| issn | 1475-2859 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj-art-fd18d2fc9f0b4611a2eedc19606755f22025-08-20T03:22:27ZengBMCMicrobial Cell Factories1475-28592025-05-0124111610.1186/s12934-025-02741-wA CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native hostZhe-Chong Wang0Hayden Stegall1Takeshi Miyazawa2Adrian T. Keatinge-Clay3Department of Molecular Biosciences, The University of Texas at AustinDepartment of Molecular Biosciences, The University of Texas at AustinDepartment of Molecular Biosciences, The University of Texas at AustinDepartment of Molecular Biosciences, The University of Texas at AustinAbstract Background Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA. Results We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin. Conclusion Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.https://doi.org/10.1186/s12934-025-02741-wPikromycinType I PKSsCRISPR/Cas9RiboswitchUpdated module boundaryModule-swapping |
| spellingShingle | Zhe-Chong Wang Hayden Stegall Takeshi Miyazawa Adrian T. Keatinge-Clay A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host Microbial Cell Factories Pikromycin Type I PKSs CRISPR/Cas9 Riboswitch Updated module boundary Module-swapping |
| title | A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host |
| title_full | A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host |
| title_fullStr | A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host |
| title_full_unstemmed | A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host |
| title_short | A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host |
| title_sort | crispr cas9 system for knock out and knock in of high molecular weight dna enables module swapping of the pikromycin synthase in its native host |
| topic | Pikromycin Type I PKSs CRISPR/Cas9 Riboswitch Updated module boundary Module-swapping |
| url | https://doi.org/10.1186/s12934-025-02741-w |
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