[Coexpression of caiB and caiE with two plasmids in E. coli]

Area of Science:

• Biotechnology
• Molecular Biology
• Enzyme Engineering

Background:

• Carnitine dehydratase (caiB) and its related cofactor synthesis protein (caiE) are crucial for carnitine metabolism.
• Efficient expression of these enzymes in a microbial host is essential for biotechnological applications.

Purpose of the Study:

• To achieve high enzymatic activity of carnitine dehydratase by coexpressing caiB and caiE genes in E. coli Bl21(DE3).
• To compare the efficacy of compatible and incompatible two-plasmid systems for gene coexpression.
• To evaluate the impact of different coexpression strategies on carnitine dehydratase activity and plasmid stability.

Main Methods:

• Cloning and coexpression of caiB and caiE genes in E. coli Bl21(DE3) using both compatible and incompatible two-plasmid systems.
• Analysis of protein expression levels via SDS-PAGE after IPTG induction.
• Quantification of carnitine dehydratase activity.
• Assessment of plasmid stability under antibiotic selective pressure.

Main Results:

• Coexpression of caiB and caiE genes was successfully achieved in both plasmid systems.
• The incompatible two-plasmid system resulted in higher expression levels (39% caiB, 20% caiE) compared to the compatible system (17% caiB, 10% caiE).
• Carnitine dehydratase activity was approximately 2.3 times higher in E. coli Bl21(DE3) with either coexpression system than with only pET28-caiB.
• Plasmid stability in both systems required antibiotic selection.

Conclusions:

• Coexpression of caiB and caiE in E. coli Bl21(DE3) enhances carnitine dehydratase activity.
• The incompatible two-plasmid system is more effective for achieving high-level coexpression of these genes.
• Further research may optimize expression and stability for industrial applications.