Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside

Area of Science:

• Biochemistry
• Synthetic Biology
• Metabolic Engineering

Background:

• Cascade reaction systems improve multistep enzymatic reactions by controlling metabolic flux.
• Generating fusion proteins for these systems is challenging due to limited expression of complex enzymes.

Purpose of the Study:

• To develop a novel fusion system that overcomes expression limitations using post-translational protein ligation.
• To create a split intein-mediated cascade system for efficient biocatalysis.

Main Methods:

• Utilized orthogonal split inteins (Cat and Rma DnaB) as adapters for protein ligation.
• Engineered a genetically programmable, self-assembled, and traceless split intein system.
• Assembled a multienzymatic complex for ginsenoside compound K (CK) production.

Main Results:

• Successfully assembled a soluble, active multienzymatic complex (>240 kDa) via split intein ligation.
• The system significantly increased CK conversion rates.
• Reduced CK production time by over 2-fold compared to traditional methods.

Conclusions:

• The split intein cascade system provides a versatile platform for regulating multimeric bioconversion pathways.
• This approach enhances the production of high-value substances like ginsenoside CK.
• The system bypasses limitations associated with complex enzyme expression in fusion proteins.