Abstract
Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100, 100, 83, and 75%, respectively, as well as the 100% editing efficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knockout five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful tool for multiplex genome editing and greatly accelerating the unraveling of hidden antibiotics in Bacillus and Paenibacillus species.
Highlights
The emergence and prevalence of multidrug-resistant bacteria (MDR) are a global health threat that causes ~700,000 deaths every year (Kollef et al, 2017)
Indicator strains obtained from the American Type Culture Collection (ATCC), the Korean Collection for Type Cultures (KCTC), or the Korean Agricultural Culture Collection (KACC) for the antimicrobial activity assay were grown as follows: Bacillus cereus ATCC 4342TM was grown in LB broth or LB agar at 30°C
Since the development of cytosine base editor (CBE) system in eukaryotes (Komor et al, 2016), base editors have been gradually applied to prokaryotes such as E. coli (Banno et al, 2018), P. aeruginosa (Chen et al, 2018), S. aureus (Gu et al, 2018), Streptomyces (Tong et al, 2019), Corynebacterium glutamicum (Wang et al, 2018), and B. subtilis (Yu et al, 2020)
Summary
The emergence and prevalence of multidrug-resistant bacteria (MDR) are a global health threat that causes ~700,000 deaths every year (Kollef et al, 2017). A process termed “dereplication” for the elimination of known antibiotics in microbial extracts has been used to resolve the issue of frequent re-isolation of the same known compound in antibiotic discovery. Dereplication has involved the use of analytical methods such as mass spectrometry, NMR-based metabolomics, and transcription profiling. These methods often require pure fractionations and are not suitable for initial screening approaches (Gaudêncio and Pereira, 2015; Genilloud, 2019). A new platform not requiring a complex purification process is warranted for rapid dereplication in microbial strains producing multiple antibiotics for efficient screening and analysis of masked compounds
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