Controlled enzyme cargo loading in engineered bacterial microcompartment shells.

Area of Science:

• Biochemistry
• Synthetic Biology
• Protein Engineering

Background:

• Bacterial microcompartments (BMCs) are protein-based organelles that encapsulate enzymes, potentially improving metabolic pathway efficiency through substrate channeling and selective permeability.
• The Haliangium ochraceum (HO) BMC shell is a modular system adaptable for expressing non-native cargo outside its native host.
• Engineered protein conjugation systems, such as SpyCatcher-SpyTag, facilitate the loading of non-native cargo into BMC shells.

Purpose of the Study:

• To investigate non-native cargo loading into four HO shell variants using triose phosphate isomerase (Tpi) as a model enzyme.
• To determine the maximal loading capacity of these HO shell variants.
• To assess the activity of encapsulated Tpi and understand the impact of shell properties on enzyme function.

Main Methods:

• Expression and assembly of four Haliangium ochraceum (HO) BMC shell variants.
• Loading of non-native triose phosphate isomerase (Tpi) into the HO shell variants using SpyCatcher-SpyTag conjugation.
• Quantification of Tpi loading levels and measurement of encapsulated Tpi activity.
• Analysis of HO shell variant assembly robustness and correlation with Tpi activity.

Main Results:

• All four HO shell variants successfully loaded non-native Tpi, demonstrating the versatility of the HO shell system.
• Enzyme activity was primarily dependent on the amount of Tpi loaded, rather than the predicted permeability of the shell variant.
• Simpler HO shell variants exhibited more robust assembly compared to more complex variants.
• Active, Tpi-loaded BMCs were generated across all tested shell variants.

Conclusions:

• The Haliangium ochraceum (HO) shell is a viable platform for engineering metabolic pathways by encapsulating non-native enzymes.
• Maximal shell loading and enzyme activity are key factors, with shell permeability having a lesser impact on large molecule encapsulation.
• The simplest HO shell variant demonstrates the most robust assembly and is recommended for future metabolic engineering applications.
• This study provides insights into optimizing BMCs for enhanced biocatalysis and metabolic engineering efforts.