A Light-Powered In Vitro Synthetic Enzymatic Biosystem for the Synthesis of 3-Hydroxypropionic Acid via CO₂ Fixation

3-Hydroxypropionic acid (3-HP) is a highly sought-after platform chemical serving as a precursor to a variety of high value-added chemical products. In this study, we designed and constructed a novel light-powered in vitro synthetic enzymatic biosystem comprising acetyl-CoA ligase, acetyl-CoA carboxylase, malonyl-CoA reductase, and phosphotransferase to efficiently produce 3-HP through CO₂ fixation from acetate, a cost-effective and readily available substrate. The system employed natural thylakoid membranes (TMs) for the regeneration of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate. Comprehensive investigations were conducted on the effects of buffer solutions, substrate concentrations, enzyme loading levels, and TMs loading levels to optimize the yield of 3-HP. Following optimization, a production of 0.46 mM 3-HP was achieved within 6 h from an initial 0.5 mM acetate, with a yield nearing 92%. This work underscores the simplicity of 3-HP production via an in vitro biomanufacturing platform and highlights the potential for incorporating TMs as a sustainable and environmentally friendly approach in biomanufacturing processes.