Phage PL-1 endolysin and osmotic stress as tools to enhance heterologous protein display in lactic acid bacteria...

Phage-based display systems using cell wall-binding domains (CBDs) from endolysins are powerful tools for anchoring proteins on bacterial surfaces. This study evaluates the CBD of the PL-1 phage endolysin (CBDLys) as a novel anchoring platform for heterologous protein display in non-genetically modified lactic acid bacteria (LAB). The system was tested by fusing CBDLys to a fluorescent reporter protein, with Lacticaseibacillus paracasei ATCC 27092 showing the highest binding capacity. Each bacterial cell bound approximately 7.04 × 10⁶ GFP-CBDLys molecules, demonstrating strong anchoring potential. To enhance binding while maintaining cell viability, we investigated pre-growth in high-salt conditions, which results in osmotic stress. This strategy significantly increased CBD-mediated binding, correlating with structural modifications in the bacterial envelope, including reduced lipoteichoic acid and polysaccharide content, as well as decreased peptidoglycan O-acetylation. These changes likely improve accessibility to PG-binding sites, optimizing CBDLys anchoring efficiency. Our findings demonstrate that osmotic stress adaptation represents a safe and effective strategy to enhance non-covalent surface display in LAB, maintaining probiotic viability and non-GMO status while improving protein presentation. The combination of the CBDLys anchoring platform with osmotic stress offers an innovative approach for probiotic-based biotechnological applications, including vaccine delivery and therapeutic protein display.