(130d) Metabolic Engineering of Clostridium Tyrobutyricum for Enhancing the Yield of Butyrate Production with Acetate Assimilation

Butyric acid, a C4 platform chemical, has a wide range of applications in the food, pharmaceutical, and animal feed industries. Recently, there has been increasing interest in the production of bio-based butyric acid, as the world is actively seeking efficient alternatives to petroleum-based products due to the global crises of climate change and fossil fuel depletion. Clostridium tyrobutyricum is a more promising native butyric acid productor. However, the yield of butyric acid is limited by the low-utilization of carbon atom due to approximately 45% carbon flow loss resulting from CO₂ and acetic acid formation. Previous research indicated that Clostridium tyrobutyricum exhibits potential for synergistically utilizing glucose and acetate to produce butyric acid. While, developing an integrated fermentation pattern, which combines the process of C. tyrobutyricum's collaborative utilization of glucose and acetate for butyric acid production with the process of fixing CO₂ to acetate by autotrophic microorganism, would break through the limitation in low-utilization of carbon atom. Therefore, our study aimed to enhance acetate assimilation in butyrate biosynthesis process. Firstly, we hypothesized that increasing intracellular NAD(P)H pool could drive acetate assimilation as additional electron acceptor. In our study, C. tyrobutyricum mutant strains overexpressing fnr, fdh, hbd and nfnAB were constructed and evaluated for their performance in silico and in vivo. One engineered C. tyrobutyricum strain significantly increased the NADH/ADH⁺ ratio intracellular, resulting in the assimilation of ~10 g/L of acetate in serum bottle fermentation and a 20.6% improvement in the yield of butyrate compared to the WT strain (0.41 ± 0.00 vs. 0.34 ± 0.01 g/g) during bioreactor-scale experiments. This study provides a foundation for further investigation of acetate assimilation and the relationship between the cofactor-redox couple and metabolic characteristics.