This study adapted an established CRISPR/Cas9 approach for the targeted disruption of plnD, a key negative regulatory gene within the plantaricin quorum-sensing network of Lactiplantibacillus plantarum 8P-A3 through extensive optimization of transformation and genome-editing conditions.
Abstract
Efficient genetic engineering of lactic acid bacteria remains technically challenging due to their thick peptidoglycan cell wall, low transformation efficiency, strain-specific restriction–modification systems, and sensitivity to Cas9-induced double-strand breaks. In this study, we adapted an established CRISPR/Cas9 approach for the targeted disruption of plnD, a key negative regulatory gene within the plantaricin quorum-sensing network of Lactiplantibacillus plantarum 8P-A3 through extensive optimization of transformation and genome-editing conditions. The genetically modified strain exhibited upregulation of plnA, plnE, and plnF, accompanied by elevated antimicrobial activity. These findings underscore the feasibility of rationally reconfiguring a quorum-sensing-associated regulatory circuit and provide a practical strategy for successful genetic engineering in L. plantarum for elevated bacteriocin production.
Where genetic accessibility can be established, the integration of CRISPR technology with synthetic biology may enable more precise gene regulation and could support the development of next-generation engineered Bifidobacterium-based platforms.
Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.
Maximilian Hilkmann, Norma Welsch, M. F. Felle et al.· Applied Microbiology and Bio...· 0 citations
Streptomyces mobaraensis is an industrially important actinomycete capable of producing transglutaminase (TGase), a valuable crosslinking enzyme that is widely used in the food, pharmaceutical, and textile industries. However, its genetic manipulation remains challenging owing to the lack of efficient genome-editing tools. Here, we characterized an endogenous type I-E CRISPR-Cas system in S. mobaraensis IPIO2 through bioinformatics analysis and plasmid interference assays, identifying the protospacer adjacent motif as 5′-AAC-3′. We engineered an artificial editing plasmid, pCRISPR, by inserting a mini-CRISPR array (repeat-spacer-repeat) and homologous recombination repair templates into the replicative plasmid pJTU1278. This system exhibited high editing efficiencies, achieving 70% for single-gene deletions and 75–80% for large DNA fragment deletions ranging from 10 to 40 kb. Based on this system, deletion of four genes consistently downregulated during TGase production, identified through comparative proteomics, enhanced TGase production by 8.5–18.5%. Furthermore, deleting the pseudouridimycin and piericidin A1 biosynthetic gene clusters using this system significantly improved the safety profile of TGase production, resulting in a 17% increase in TGase yield. This study established a robust and efficient endogenous CRISPR-Cas-based genome-editing platform in S. mobaraensis, providing a powerful tool for strain engineering and industrial optimization of TGase production.
Dan Wang, Meng-Jun Sun, Qing-Xu Meng et al.· Engineering Microbiology· 0 citations
This chapter presents a step-by-step protocol for designing sgRNAs, constructing CRISPRi plasmids, transforming F. nucleatum ATCC 23726, and evaluating gene silencing phenotypes, using the nonessential gene ftsW, which encodes a protein required for peptidoglycan synthesis and cell division, as a model target.
Shiqi Xu, B. C., Kexin Tan et al.· Methods in molecular biology· 0 citations
This chapter describes strategies to evade RM defenses and improve transformation efficiencies across diverse Fusobacterium lineages, employing a sequence-based "RM-silencing" method that has enabled successful delivery of replicative plasmids, linear recombination templates, and transposon cassettes into previously intractable Fusobacterium clinical isolates.
Martha A. Zepeda-Rivera, Elsa F. McMahon, Kaitlyn N. Lewis et al.· Methods in molecular biology· 0 citations
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