# Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene

## TL;DR

Researchers developed a new genome editing method to inactivate a hemolysin gene in two probiotic bacteria, reducing their hemolytic activity.

## Contribution

A novel ribonucleoprotein-mediated editing system was developed for Lactiplantibacillus plantarum and Levilactobacillus brevis with reduced reliance on plasmids and antibiotics.

## Key findings

- The hlyIII gene was successfully edited with a 50 bp deletion and a stop codon in all tested strains.
- Hemolytic activity was reduced in ΔhlyIII mutants compared to wild-type strains.
- Strain-dependent differences in hemolytic activity were observed, suggesting functional variability.

## Abstract

Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 − 1 and L. brevis SPC-SNU 70 − 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 − 1, and L. brevis SPC-SNU 70 − 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.