Thermodynamically Coupled Three-Enzyme Cascade Converts Styrene to Cinnamic Acid, <scp>l</scp>-Phenylalanine, and Phenylpyruvate via CO₂ Fixation without External Energy Cofactors

We report the development of a cofactor-free CO₂ fixation platform based on a three-enzyme cascade comprising <i>Aspergillus niger</i> ferulic acid decarboxylase (AnFDC), <i>Anabaena variabilis</i> phenylalanine ammonia-lyase (AvPAL), and <i>Proteus mirabilis</i> l-amino acid deaminase (PmLAAD). Unlike canonical ATP- or NADPH-dependent CO₂ assimilation pathways, this system uses a prFMN-dependent carboxylation mechanism, enabling efficient CO₂ incorporation under ambient conditions without energy-intensive cofactors. Systematic screening identified AnFDC as the optimal decarboxylase for styrene carboxylation, while AvPAL and PmLAAD were selected for their superior catalytic efficiencies in the cascade. Optimization of prFMN biosynthesis (via UbiX/SccK coexpression), enzyme expression, and reaction conditions (notably, 1.5 M ammonium bicarbonate, pH 8.5) significantly enhanced cascade performance. Whole-cell microbial consortia with tailored cell ratios further alleviated kinetic bottlenecks, achieving a 3-fold improvement in phenylpyruvic acid production (6% conversion) from styrene and CO₂. The integrated cascade drives the CO₂ fixation with an overall equilibrium constant (K_eq') of 4.3 × 10³⁰, converting low-cost styrene into high-value phenylpyruvic acid. Through enzyme screening and step-by-step optimization, we established an energy-independent system for CO₂ fixation. Our work challenges the cofactor dependence in biocatalytic carbon fixation for aromatic compounds and paves a novel way for sustainable, carbon-negative chemical manufacturing.