Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Abstract

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48h) anaerobic reaction in metabolically engineered strains. At 42°C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30°C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36°C, 28% higher than that at 30°C, although the production at 42°C was still 23% higher than that at 30°C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48h at 42°C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.