Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Lactic acid bacteria (LAB) are crucial in food, feed, and health industries.
• Traditional genome manipulation in LAB has been slow and inefficient.
• Previous methods like homologous recombination and transposon mutagenesis had limitations.

Purpose of the Study:

• To review the evolution of genome editing strategies in lactic acid bacteria.
• To highlight advancements from early methods to modern CRISPR-based systems.
• To discuss the potential of these tools for LAB biotechnology.

Main Methods:

• Evolutionary review of genome editing techniques in LAB.
• Comparison of RecA-dependent double-crossovers, recombineering, and CRISPR systems.
• Analysis of phage-derived enzymes and CRISPR-associated transposases.

Main Results:

• Early methods (homologous recombination, transposon mutagenesis) offered targeted gene disruption but with low efficiency and marker issues.
• Modern phage-mediated recombineering and CRISPR/Cas counter-selection enable scar-less edits, deletions, and large insertions with high efficiency.
• CRISPR tools allow multiplex knockouts, precise mutations, and conditional gene expression.

Conclusions:

• Advanced genome editing tools are transforming LAB into versatile platforms for industrial applications.
• CRISPR and recombineering significantly enhance the precision and efficiency of LAB genetic engineering.
• Overcoming strain-specific barriers and improving tool host-range are key for future development.