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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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Autores principales: Hu, Mengyi, Chen, Shuo, Ni, Yao, Wei, Wei, Mao, Wenwei, Ge, Mei, Qian, Xiuping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10020181/
https://www.ncbi.nlm.nih.gov/pubmed/36937767
http://dx.doi.org/10.3389/fbioe.2023.1141176
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author Hu, Mengyi
Chen, Shuo
Ni, Yao
Wei, Wei
Mao, Wenwei
Ge, Mei
Qian, Xiuping
author_facet Hu, Mengyi
Chen, Shuo
Ni, Yao
Wei, Wei
Mao, Wenwei
Ge, Mei
Qian, Xiuping
author_sort Hu, Mengyi
collection PubMed
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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spelling pubmed-100201812023-03-18 CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila Hu, Mengyi Chen, Shuo Ni, Yao Wei, Wei Mao, Wenwei Ge, Mei Qian, Xiuping Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2023-03-03 /pmc/articles/PMC10020181/ /pubmed/36937767 http://dx.doi.org/10.3389/fbioe.2023.1141176 Text en Copyright © 2023 Hu, Chen, Ni, Wei, Mao, Ge and Qian. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Hu, Mengyi
Chen, Shuo
Ni, Yao
Wei, Wei
Mao, Wenwei
Ge, Mei
Qian, Xiuping
CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila
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 Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10020181/
https://www.ncbi.nlm.nih.gov/pubmed/36937767
http://dx.doi.org/10.3389/fbioe.2023.1141176
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