Rational design of a bacterial import system for new-to-nature molecules

Area of Science:

• Synthetic biology
• Biochemistry
• Molecular biology

Background:

• Cellular uptake of novel compounds is crucial for expanding biological functions.
• Selectively permeable cell membranes pose a significant barrier to compound entry.
• Existing methods for compound delivery are often inefficient or limited in scope.

Purpose of the Study:

• To design and validate a novel bacterial transport system for enhanced cellular uptake of impermeant molecules.
• To overcome the limitations of natural cell membrane permeability using synthetic biology approaches.
• To demonstrate the broad applicability and in vivo functionality of the engineered system.

Main Methods:

• Rational design of a bacterial transport system utilizing a promiscuous sulfonate membrane transporter.
• Attachment of cargo molecules to a sulfobutanoate transport vector via amide linkage.
• Enzymatic cleavage of the amide bond using an engineered γ-glutamyl transferase variant for cargo release.
• Evaluation of substrate specificity using diverse sulfobutanoate amides.
• In vivo implementation and functional assessment in a bacterial system.

Main Results:

• Demonstrated broad substrate specificity for both the sulfonate transporter and the engineered γ-glutamyl transferase.
• Successfully imported structurally diverse cargo molecules into the bacterial cytoplasm.
• Validated the system's functionality in vivo by importing an impermeant non-canonical amino acid.
• Confirmed efficient release of cargo molecules within the cell following transport.

Conclusions:

• The engineered bacterial transport system effectively overcomes cell membrane barriers for compound delivery.
• The system exhibits broad substrate specificity, enabling the import of various cargo molecules.
• This novel approach provides a powerful tool for synthetic biology, drug delivery, and metabolic engineering applications.
• The in vivo functionality highlights the potential for therapeutic and biotechnological applications.