De novo synthesis of p-coumaric acid from fructose and carbon dioxide in chemoautotrophic Cupriavidus necator H16

p-Coumaric acid (p-CA) is a valuable phenolic compound widely applied in food, pharmaceutical, and cosmetic industries. While the chemolithoautotrophic Cupriavidus necator H16 is a potent host for converting CO₂ into biochemicals, its potential for synthesizing aromatic-derived compounds remains to be fully explored. In this study, p-CA was selected as a model compound to systematically engineer the metabolic network of C. necator H16 for aromatic biosynthesis from fructose and CO₂. We first established a tyrosine-derived pathway and subsequently enhanced the metabolic flux by identifying and overexpressing key pathway genes—aroG1, aroQ1, and aroC. Then, the carbon flux was redirected towards tyrosine by replacing the native prephenate dehydratase (PheA) with Escherichia coli prephenate dehydrogenase (TyrA). Furthermore, we introduced the E. coli nicotinamide nucleotide transhydrogenase to increase cofactor availability and optimized the process by substituting ammonium chloride with urea. These systematic modifications resulted in an engineered strain producing 25.4 mg/L of p-CA from fructose, a 1,593.3% increase compared to the initial strain. Significantly, under autotrophic conditions, the strain enabled de novo synthesis of p-CA from CO₂, reaching 3.1 mg/L. This work not only demonstrates the first light-independent p-CA biosynthesis from CO₂ but also validates the feasibility of using C. necator H16 as a sustainable platform for the production of aromatic chemicals.