Cargando…
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...
Autores principales: | , , , , , , |
---|---|
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 |
_version_ | 1784908195541549056 |
---|---|
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. |
format | Online Article Text |
id | pubmed-10020181 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT humengyi crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT chenshuo crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT niyao crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT weiwei crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT maowenwei crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT gemei crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila AT qianxiuping crisprcas9mediatedgenomeeditinginvancomycinproducingstrainamycolatopsiskeratiniphila |