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CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila.


ABSTRACT: 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.

SUBMITTER: Hu M 

PROVIDER: S-EPMC10020181 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated genome editing in vancomycin-producing strain <i>Amycolatopsis keratiniphila</i>.

Hu Mengyi M   Chen Shuo S   Ni Yao Y   Wei Wei W   Mao Wenwei W   Ge Mei M   Qian Xiuping X  

Frontiers in bioengineering and biotechnology 20230303


<i>Amycolatopsis</i> is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some <i>Amycolatopsis</i> 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 <i>A. keratiniphila</i> HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-05  ...[more]

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