Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

• Highly selective butyric acid production was achieved in C. acetobutylicum.
• Butyric acid to acetic acid (B/A) ratio was increased by disrupting the buk gene.
• The B/A ratio was further increased by disrupting the adhE1 and hydA genes.
• Butyric acid was produced to 33 g/l with a B/A ratio of 31.
• Quintuple gene knockout of C. acetobutylicum was demonstrated for the first time.

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta−, buk−, ctfB− and adhE1−) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta−, buk−, ctfB−, adhE1− and hydA−) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.