'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria

Area of Science:

• * Microbiology and Genetic Engineering
• * Bacterial Transformation and Gene Integration
• * Metabolic Engineering

Background:

• * Established methods for genetic manipulation of Corynebacterium melassecola are limited due to host restriction systems.
• * Escherichia coli-derived plasmids face challenges when introduced into C. melassecola.
• * Brevibacterium lactofermentum serves as a useful intermediate host for plasmid modification.

Purpose of the Study:

• * To develop a method for efficient gene integration into the Corynebacterium melassecola chromosome.
• * To overcome host restriction barriers in C. melassecola.
• * To enable stable expression of heterologous genes in C. melassecola.

Main Methods:

• * Construction of a shuttle plasmid (pCGL519-2) carrying the C. melassecola gltA gene.
• * Electroporation of the shuttle plasmid into C. melassecola.
• * Isolation of an 'integron' fragment containing a selectable marker and the gltA gene.
• * Homologous recombination for chromosomal integration of the integron.

Main Results:

• * A method was established to evade C. melassecola restriction barriers using modified plasmids.
• * An integron containing the citrate synthase (gltA) gene was successfully integrated into the C. melassecola chromosome.
• * The resulting transformants exhibited stable gene expression for at least 30 generations without selection.

Conclusions:

• * The developed integron system allows for efficient and stable chromosomal integration in C. melassecola.
• * This method overcomes previous limitations associated with genetic manipulation of this bacterium.
• * The stable transformants are valuable for further metabolic engineering applications.