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

Journal of Biological Engineering, vol. 20

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.

Authors 3

1. Rudolf Lütticken Aachen DWI - Leibniz Institute for Interactive Materials

   DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI - Leibniz Institute for Interactive Materials, 52074, Aachen, Germany

2. Laura De Laporte Aachen DWI - Leibniz Institute for Interactive Materials Institute for Technical and Macromolecular Chemistry Institute of Applied Medical Engineering Department of Advanced Materials for Biomedicine

   RWTH Aachen University · Universitätsklinikum Aachen · DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI - Leibniz Institute for Interactive Materials, 52074, Aachen, Germany

   Institute for Technical and Macromolecular Chemistry, RWTH Aachen University, Aachen, Germany

   Institute of Applied Medical Engineering (AME), Department of Advanced Materials for Biomedicine (AMB), University Hospital RWTH Aachen, Center for Biohybrid Medical Systems (CMBS), Aachen, Germany

3. Elisabeth Heine corresponding Aachen DWI - Leibniz Institute for Interactive Materials

   RWTH Aachen University · DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI - Leibniz Institute for Interactive Materials, 52074, Aachen, Germany. heine@dwi.rwth-aachen.de