Parallel Bottlenecking-Debottlenecking Evolution and Promoter Reprogramming Enable Epistasis-Resilient Pinosylvin...

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biocatalysis

Background:

• Epistatic interactions between enzymes are a major challenge in metabolic pathway engineering.
• Designing complex biosynthetic pathways is often hindered by unpredictable gene-gene interactions.

Purpose of the Study:

• To develop a generalizable strategy for overcoming epistatic barriers in complex biosynthetic pathways.
• To engineer microbial pinosylvin biosynthesis by integrating evolutionary and genetic engineering approaches.

Main Methods:

• Mapped evolutionary trajectories of pinosylvin biosynthesis enzymes.
• Developed a transcription-factor-based biosensor for parallel enzyme evolution.
• Implemented combinatorial analysis and targeted promoter reprogramming.

Main Results:

• Identified prevalent gene-gene epistasis affecting metabolic flux.
• Rebalanced transcriptional flux through promoter reprogramming, restoring pathway coordination.
• Achieved 931.04 mg/L pinosylvin in fed-batch fermentation, surpassing previous systems.

Conclusions:

• Established a scalable strategy combining landscape-guided evolution, biosensor selection, and modular expression control.
• Demonstrated a generalizable approach for overcoming epistatic barriers in complex biosynthetic design.
• The engineered strain achieved high pinosylvin titers without host modifications.