Computer-Aid Directed Evolution of GPPS and PS Enzymes

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Pinene, a valuable monoterpene, has diverse applications but low microbial production yields.
• Challenges include low enzyme expression (GPPS, PS) and monoterpene cytotoxicity.
• Existing yields lag behind those of other terpenes like isoprene and farnesene.

Purpose of the Study:

• To improve the biological yield of pinene in genetically engineered microorganisms.
• To overcome limitations in enzyme expression and product toxicity.

Main Methods:

• Deletion of random coils in geranyl pyrophosphate synthase (GPPS) and pinene synthase (PS).
• Computer-guided molecular modeling and docking to predict key GPPS active sites.
• Site-directed mutagenesis of GPPS, focusing on substrate-binding regions.

Main Results:

• A 335% increase in pinene yield was achieved by deleting random coils in GPPS and PS.
• Molecular modeling identified critical residues in GPPS for substrate (IPP) binding.
• A T273R mutation in GPPS resulted in a 154% increase in pinene yield.

Conclusions:

• Enzyme engineering, specifically coil deletion and targeted mutagenesis, significantly enhances microbial pinene production.
• Strategies address both enzyme efficiency and potential product toxicity.
• This work provides a foundation for scalable biotechnological production of pinene.