The Chromosomal Gene Manipulation Method for Lactobacillus delbrueckii subsp. bulgaricus Using a Conjugative Shuttle...

Area of Science:

• Microbiology
• Molecular Biology
• Industrial Biotechnology

Background:

• Lactobacillus bulgaricus is crucial for dairy fermentation but challenging to genetically modify.
• Limited tools exist for chromosomal gene manipulation (deletion, insertion, replacement) in L. bulgaricus.

Purpose of the Study:

• To develop an efficient system for chromosomal gene manipulation in L. bulgaricus.
• To demonstrate the utility of this system by deleting a gene involved in extracellular polysaccharide synthesis.

Main Methods:

• Development of a conjugal transfer vector system for easy vector construction in E. coli.
• Detailed protocols for vector construction, conjugation, and double recombination to obtain deletion strains.
• Utilized the developed system to delete a specific regulatory gene in L. bulgaricus.

Main Results:

• Successfully established a system for chromosomal gene manipulation in L. bulgaricus.
• Demonstrated gene deletion capability by removing a regulatory gene for extracellular polysaccharide synthesis.
• Provided detailed methods for vector construction and strain generation.

Conclusions:

• The developed conjugal transfer vector system significantly simplifies genetic manipulation of L. bulgaricus.
• This system facilitates functional genomic studies, such as elucidating gene functions related to polysaccharide synthesis.
• The vector pGMβ1 is available via Addgene for academic research, promoting further advancements.