CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila
Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRIS...
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1141176/full |
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| author | Mengyi Hu Shuo Chen Yao Ni Wei Wei Wenwei Mao Mei Ge Xiuping Qian |
| author_facet | Mengyi Hu Shuo Chen Yao Ni Wei Wei Wenwei Mao Mei Ge Xiuping Qian |
| author_sort | Mengyi Hu |
| collection | DOAJ |
| description | Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%–97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%–40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species. |
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| language | English |
| last_indexed | 2024-04-10T06:03:24Z |
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| spelling | doaj.art-00be6ed8d9724a69ae2ff9d02805f3ec2023-03-03T04:35:56ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-03-011110.3389/fbioe.2023.11411761141176CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphilaMengyi Hu0Shuo Chen1Yao Ni2Wei Wei3Wenwei Mao4Mei Ge5Xiuping Qian6School of Pharmacy, Shanghai Jiao Tong University, Shanghai, ChinaSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, ChinaSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, ChinaShanghai Laiyi Center for Biopharmaceutical R&D, Shanghai, ChinaSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, ChinaShanghai Laiyi Center for Biopharmaceutical R&D, Shanghai, ChinaSchool of Pharmacy, Shanghai Jiao Tong University, Shanghai, ChinaAmycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%–97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%–40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1141176/fullamycolatopsisCRISPR/Cas9genome editinglarge fragment deletionEco-0501vancomycin |
| spellingShingle | Mengyi Hu Shuo Chen Yao Ni Wei Wei Wenwei Mao Mei Ge Xiuping Qian CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila Frontiers in Bioengineering and Biotechnology amycolatopsis CRISPR/Cas9 genome editing large fragment deletion Eco-0501 vancomycin |
| title | CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila |
| title_full | CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila |
| title_fullStr | CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila |
| title_full_unstemmed | CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila |
| title_short | CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila |
| title_sort | crispr cas9 mediated genome editing in vancomycin producing strain amycolatopsis keratiniphila |
| topic | amycolatopsis CRISPR/Cas9 genome editing large fragment deletion Eco-0501 vancomycin |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1141176/full |
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