AUTHOR=Hu Mengyi , Chen Shuo , Ni Yao , Wei Wei , Mao Wenwei , Ge Mei , Qian Xiuping 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1141176

DOI=10.3389/fbioe.2023.1141176

ISSN=2296-4185

ABSTRACT=<p><italic>Amycolatopsis</italic> is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some <italic>Amycolatopsis</italic> 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 <italic>A. keratiniphila</italic> HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of <italic>gapdh</italic>p and <italic>ermE*</italic>p which drove the expressions of <italic>scocas9</italic> and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene <italic>gtfD</italic> and inserted the gene <italic>eGFP</italic> with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of <italic>cds13-17</italic> (7.7 kb), <italic>cds23</italic> (12.7 kb) and <italic>cds22-23</italic> (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 <italic>cds4-27</italic> (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 <italic>cds13-17</italic>, <italic>cds22-23</italic> and <italic>cds4-27</italic>, all achieved a 30%–40% increase in vancomycin yield<italic>.</italic> Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in <italic>A. keratiniphila</italic> HCCB10007 might provide a powerful tool for other <italic>Amycolatopsis</italic> species.</p>