An optimized CRISPR/Cas9-based genome editing platform enhances bacteriocin production in Lactiplantibacillus plantarum

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.