Progressive probing and iterative integration enable strain-state-specific fine-tuning of the MEP pathway for patchoulol biosynthesis in Bacillus subtilis

Background Efficient optimization of metabolic pathways in microbial cell factories remains challenging because the relative contributions of individual pathway enzymes can change with the metabolic state of engineered strains. Conventional metabolic engineering approaches generally rely on predefined targets or simultaneous overexpression of multiple pathway genes, which may lead to pathway imbalance and limited improvement. Here, we developed a Progressive Probing and Iterative Integration (PP&II) strategy for successive and strain-state-specific optimization of metabolic pathways. Results PP&II integrates a reusable enzyme-probing module with a CRISPR-Cas9-assisted genomic integration module, enabling assessment of enzyme contributions in each engineered strain and targeted genomic integration in successive cycles. After each engineering cycle, enzyme contributions were reassessed to guide the next round of genomic integration. The PP&II strategy was validated in the generally recognized as safe (GRAS) host Bacillus subtilis using patchoulol biosynthesis as a model system. Following establishment of a functional patchoulol biosynthetic pathway through optimization of heterologous patchoulol synthase expression, PP&II enabled successive assessment and genomic optimization of multiple 2C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes, including Dxs, IspC, and IspA. The optimized strain produced approximately 13-fold more patchoulol than the initial strain. Comparative proteomic analysis further revealed changes in protein expression following PP&II-based engineering, indicating alterations in the cellular state of the engineered strain. Conclusions Overall, PP&II provides a progressive, strain-state-guided workflow for iterative engineering of metabolic pathways and shows potential for improving microbial production of high-value terpenoids and other bioproducts.