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

is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some 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 HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of p and p which drove the expressions of and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene and inserted the gene with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of (7.7 kb), (12.7 kb) and (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 (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 , and , 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 HCCB10007 might provide a powerful tool for other species.