Core Viewpoint - Lignin valorization is crucial for achieving economically viable and sustainable lignocellulosic biorefineries, with a focus on overcoming the challenges posed by expensive cofactors and enzyme activity imbalance [1][3][14] Group 1: Lignin Valorization Challenges - The continuous demand for expensive cofactors like ATP, NADPH, and CoA significantly hinders the sustainable conversion of lignin into valuable products [1] - The production rates of valuable molecules derived from lignin, such as raspberry ketone (RK) and curcumin, are extremely low, highlighting the inefficiencies in current conversion processes [1][3] Group 2: iMECS Strategy Development - The iMECS (in vitro multi-enzyme coordinated expression with cofactor self-circulation) strategy integrates cofactor regeneration and cell-free expression into the lignin upgrading process [3][4] - The iMECS platform demonstrated a 48-fold increase in overall catalytic efficiency without the addition of external cofactors, achieving a curcumin production rate of 0.175 g/L/h and a molar yield of 95.31% [3][6] Group 3: Multi-Enzyme Optimization - The study optimized the production of curcumin by establishing a biocatalytic module and introducing an in situ multi-cofactor recovery module, resulting in a curcumin yield of 126.4 mg/L [5][6] - The iMECS system achieved a curcumin production rate that was over 1455% higher than traditional whole-cell bioconversion methods [6] Group 4: Application to Vanillin Synthesis - The iMECS system was further evaluated for the conversion of ferulic acid to vanillin, achieving a vanillin yield of 94.4 mg/L with a 67% reduction in ATP demand [7][8] - The system's efficiency was enhanced by employing a temperature-guided biocatalytic process, which maintained enzyme activity while inactivating undesirable endogenous enzymes [9] Group 5: RK Biosynthesis - The iMECS strategy was applied to the more complex pathway of converting p-coumaric acid (pCA) to RK, achieving a production rate of 0.15 g/L/h and a conversion rate of 91.56% [10][13] - The integration of upstream and downstream modules within the iMECS system demonstrated significant potential for synthesizing more complex products from lignin-derived substrates [12][13] Group 6: Environmental and Economic Implications - The iMECS strategy promotes the use of lignin-rich agricultural waste as a feedstock, decoupling biomanufacturing from sugar prices and reducing greenhouse gas emissions compared to traditional petroleum-based synthesis [14]