To alleviate carbon dioxide (CO₂) emission, alternative approaches have been considered, such as employing microorganisms capable of CO₂-fixation using their own metabolic pathways.

One of the current promising solutions to address problems of CO ₂ emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO ₂ fixation. In a previous study, we introduced the Calvin-Benson Bassham (CBB) genes of the photosynthetic bacterium Cereibacter sphaeroides into Escherichia coli to develop a strain capable of endogenous CO ₂ recycling by heterologous expression of the CBB genes. However, the heterologous expression of recombinant proteins in E. coli is often hampered by inclusion bodies (IB), which are protein aggregates. Various factors contribute to IB formation, including host cell metabolism, protein synthesis, transformation machinery, and target protein properties. In this study, we investigated the influence of environmental conditions, particularly culture temperature, on IB formation and CO ₂ recycling in the CBB strain. As a result, by reducing the culture temperature from 37°C to 30°C, a significant suppression of IB formation was achieved, resulting in a remarkable decrease in CO ₂ release by approximately 5.76 times. In addition, interestingly, an enhancement in the accumulation of pyruvate by approximately 2.3 times was observed at the same time. These results demonstrate the simultaneous improvements in CO ₂ recycling and the synthesis of organic acids achieved through temperature control. Based on the findings of this study, we believe that temperature control would be a promising approach to increasing chemical biosynthetic productivity as well as biological CO ₂ fixation activity in integrated autotrophic biorefinery strategies. IMPORTANCE: In a previous study, we successfully engineered Escherichia coli capable of endogenous CO ₂ recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO ₂ fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO ₂ recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO ₂ recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.