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

Epistatic interactions among heterologous enzymes remain a fundamental bottleneck in pathway engineering, often constraining accessible evolutionary trajectories and frustrating rational pathway design. Using microbial pinosylvin biosynthesis as a model, we first mapped the controllable evolutionary trajectories for each pinosylvin-associated enzyme. We then developed a transcription-factor-based biosensor that links target product accumulation to fluorescence output, enabling the parallel evolution of all core enzymes along their manageable trajectories. A subsequent combinatorial analysis revealed the prevalent gene-gene epistasis, demonstrating that continued parallel evolution of all pinosylvin-related enzymes might compromise metabolic flux by creating conflicting mutational effects. To circumvent this, we further implemented targeted promoter reprogramming to rebalance transcriptional flux, restoring pathway coordination and unlocking synergistic improvements across evolved modules. The optimal strain achieved 931.04 mg/L pinosylvin in fed-batch fermentation, exceeding previously reported systems without requiring host-level changes. These results establish a scalable strategy that integrates landscape-guided evolution, biosensor-driven selection, and modular expression control, offering a generalizable approach for overcoming epistatic barriers in complex biosynthetic design.