Engineering <i>Halomonas bluephagenesis</i> for Efficient Production of Poly-3-hydroxybutyrate Bioplastics Using CO₂-Derived Ethanol

Engineering microbial cell factories to convert CO2 into high-value chemicals represents a promising avenue toward achieving carbon neutrality and sustainable development. Halomonas bluephagenesis, a halophilic bacterium exhibiting a rapid growth rate in saline and alkaline environments, has been developed for cost-effective production of poly-3-hydroxybutyrate (PHB), ectoine, and other block chemicals. Here, we first investigated the natural ethanol assimilation pathways of H. bluephagenesis and developed the strategies to synthesize PHB using CO2-derived ethanol as the carbon source. Ethanol degradation route and crucial metabolic targets were elucidated through transcriptome analysis. Substrate utilization was further improved through screening and precise tuning of the expression level of key enzymes. Subsequently, by modulating the chromosomal expression of alcohol dehydrogenase and acetaldehyde acetylating dehydrogenase to balance the carbon flow, an engineered H. bluephagenesis was constructed, achieving a PHB titer of 4.24 g/L using ethanol in shake flask cultures. Finally, Clostridium ragsdalei was employed to convert CO2 and H2 into ethanol, then ethanol was utilized by H. bluephagenesis to generate PHB, reaching a PHB titer of 64.89 g/L. This study demonstrates a promising closed-loop process to synthesize biodegradable plastics from CO2.