Increase CO₂ recycling of Escherichia coli containing CBB genes by enhancing solubility of multiple expressed proteins from an operon through temperature reduction

One of the current promising solutions to address problems of CO₂ emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 CO₂ fixation activity in integrated autotrophic biorefinery strategies.