Display of functionally active PHB depolymerase on Escherichia coli cell surface

Area of Science:

• Biotechnology and Metabolic Engineering
• Enzyme Engineering and Biocatalysis
• Polymer Science and Engineering

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with diverse applications.
• Efficient enzymatic degradation of PHAs into valuable monomers is crucial for sustainable production.
• Cell surface display technology offers a promising platform for whole-cell biocatalysts.

Purpose of the Study:

• To engineer Escherichia coli (E. coli) for surface display of polyhydroxyalkanoate (PHA) depolymerase (PhaZ(RpiT1)).
• To evaluate the enzymatic activity and stability of the displayed PHA depolymerase.
• To assess the potential of engineered E. coli as a whole-cell biocatalyst for PHA degradation.

Main Methods:

• Construction of a fusion protein between PhaZ(RpiT1) and OprI from Pseudomonas aeruginosa for cell surface anchoring.
• Stable expression and surface localization of the fusion protein in E. coli JM109, verified by immunofluorescence microscopy.
• Assessment of the catalytic activity of displayed PhaZ(RpiT1) on both soluble and insoluble PHA substrates.

Main Results:

• Successful display of functional PhaZ(RpiT1) on the surface of E. coli JM109 cells.
• The displayed enzyme retained its ability to cleave soluble substrates and adsorb to PHA surfaces.
• Catalytic activity was maintained for the degradation of insoluble PHA materials, demonstrating potential as a whole-cell biocatalyst.

Conclusions:

• Engineered E. coli displaying PhaZ(RpiT1) serves as an effective whole-cell biocatalyst for PHA degradation.
• This approach facilitates the production of (R)-3-hydroxybutyrate monomers from insoluble PHA.
• Cell surface display of PHA depolymerase offers a sustainable and efficient method for PHA valorization.