Bioengineering of bacteria to assemble custom-made polyester affinity resins

Area of Science:

• Biotechnology
• Materials Science
• Synthetic Biology

Background:

• Traditional protein purification methods can be complex and costly.
• Developing novel affinity resins for bioseparation is crucial for advancing biotechnology.
• Microbial production offers a sustainable and scalable alternative for biomaterial synthesis.

Purpose of the Study:

• To demonstrate the in vivo bacterial production of polyester resins with customizable protein-binding domains.
• To engineer bacterial polyester synthases for the incorporation of various affinity domains.
• To establish a proof-of-concept for microbial synthesis of bioseparation resins.

Main Methods:

• Engineering bacterial polyester-synthesizing enzymes to display affinity domains.
• Utilizing designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains (nanobodies).
• Producing resins via single-step bacterial fermentation and developing simple purification protocols.

Main Results:

• Successfully produced polyester resins with specific affinity for target proteins.
• Achieved binding capacities ranging from 90 to 600 nmol/g wet resin.
• Enabled one-step purification of recombinant proteins to 96% purity from bacterial lysates.
• Demonstrated intracellular coproduction and purification of target proteins within bacteria.

Conclusions:

• Microbial production of polyester affinity resins is feasible and efficient.
• Engineered polyester synthases can be used to create customizable bioseparation materials.
• This technique holds promise for scalable and cost-effective protein purification.