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Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

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Cargo encapsulation in bacterial microcompartments: Methods and analysis.

Taylor M Nichols¹, Nolan W Kennedy², Danielle Tullman-Ercek³

¹Department of Chemical and Biological Engineering, Northwestern University, Technological Institute, Evanston, IL, United States.

Methods in Enzymology

|February 21, 2019

Summary

This summary is machine-generated.

Metabolic engineering uses bacterial microcompartments (MCPs) as scaffolds to organize cellular pathways. This approach enhances production of valuable compounds by improving enzyme efficiency and managing toxic intermediates.

Keywords:

Bacterial microcompartments (MCPs)Encapsulation Enzyme assays Flow cytometry Fluorescence microscopy Metabolic engineering Microcompartment purification Protein scaffolds Salmonella enterica serovar Typhimurium LT2 Targeting sequences

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Area of Science:

Biotechnology
Synthetic Biology
Metabolic Engineering

Background:

Metabolic engineering aims to produce valuable compounds using microbes, but faces challenges like pathway bottlenecks and toxicity.
Pathway organization using scaffolds, particularly within subcellular compartments, offers a promising solution.
Scaffolding increases local concentrations, sequesters toxic intermediates, and reduces resource competition.

Purpose of the Study:

To describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as a scaffold for heterologous pathway organization.
To present methods for controlling Pdu MCP formation, cargo expression, and loading.
To introduce assays for analyzing Pdu MCPs and their encapsulation efficiency.

Main Methods:

Utilizing the Pdu MCP system for encapsulating heterologous metabolic pathways.
Developing protocols for controlling MCP formation and heterologous cargo expression.
Implementing assays to quantify cargo loading and analyze MCP structure.

Main Results:

Demonstrated successful encapsulation of heterologous pathways within Pdu MCPs.
Established methods for controlling MCP formation and cargo loading levels.
Validated assays for assessing MCP encapsulation efficiency.

Conclusions:

Pdu MCPs serve as effective enclosed scaffolds for metabolic pathway sequestration and organization.
The described methods enable the repurposing of MCPs as tunable nanobioreactors.
This approach holds significant potential for optimizing metabolic engineering applications.