Food-grade delivery system for controlled gene expression in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Food Biotechnology

Background:

• Lactococcus lactis is a key bacterium in dairy fermentation.
• Efficient genetic manipulation systems are crucial for improving L. lactis strains.
• Existing methods may lack food-grade compliance or efficient gene transfer.

Purpose of the Study:

• To establish a food-grade system for gene delivery, inducible expression, and transfer in Lactococcus lactis.
• To integrate and express heterologous genes, specifically peptidase genes, in L. lactis.
• To demonstrate the transferability of engineered genetic elements between L. lactis strains.

Main Methods:

• Construction of thermosensitive plasmid vectors based on the pG(+)host replicon.
• Integration of genes into the L. lactis chromosome or sex factor via single crossovers.
• Utilized the nisin-controlled expression (NICE) system for inducible gene transcription.
• Conjugation was used to transfer engineered sex factors between L. lactis strains.

Main Results:

• Successfully constructed food-grade L. lactis recombinants with integrated signal transduction genes (nisRK) and peptidase (pep) gene fusions.
• Demonstrated inducible expression of P(nisA)::pep fusions using the NICE system after nisin induction.
• Observed variable induction rates depending on the integration locus of nisRK.
• Achieved successful transfer of an engineered sex factor carrying a P(nisA)::pepI fusion via conjugation at a frequency of 4 x 10(-4).

Conclusions:

• A versatile food-grade genetic system for L. lactis has been developed.
• The system allows for inducible expression of cloned genes and efficient gene transfer.
• This platform holds potential for metabolic engineering of L. lactis for improved food production.