Enhanced butyric acid production in Clostridium tyrobutyricum by overexpression of rate-limiting enzymes in the Embden-Meyerhof-Parnas pathway

Abstract

•Clostridium tyrobutyricum is an excellent microorganism for bio-based butyric acid production. However, the main obstacles for its industrialization are low butyric acid concentration and productivity. This study indicated that overexpression of rate-limiting enzymes in the Embden-Meyerhof-Parnas pathway, including 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA), in C. tyrobutyricum ATCC 25755 could enhance its intracellular NADH and ATP levels and resistance to butyric acid and glucose. The results of fed-batch fermentation by ATCC 25755/pfkA + pykA showed that the butyrate concentration and productivity reached 48.2 g/L and 0.50 g/L h, which increased 38.1% and 38.9% as compared with the wild-type, respectively. The increased butyric acid tolerance and production could be due to the higher intracellular NADH and ATP levels by overexpression of pfkA and pykA in C. tyrobutyricum ATCC 25755. Furthermore, ATCC 25755/pfkA+pykA exhibited higher glucose tolerance. Consequently, the productivity of butyrate was further increased by 64.0% in batch fermentation using high concentration of glucose (120 g/L).