초록
<P><B>Abstract</B></P> <P> <UL> <LI> <I>Clostridium tyrobutyricum</I> 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 (<I>pfkA</I>) and pyruvate kinase (<I>pykA</I>), in <I>C. tyrobutyricum</I> 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/<I>pfkA</I> + <I>pykA</I> 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 <I>pfkA</I> and <I>pykA</I> in <I>C. tyrobutyricum</I> ATCC 25755. Furthermore, ATCC 25755/<I>pfkA</I>+<I>pykA</I> 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). </LI> </UL> </P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>C</I>. <I>tyrobutyricum</I> was engineered to enhance the Embden-Meyerhof-Parnas pathway. </LI> <LI> Overexpression of <I>pfkA</I> and/or <I>pykA</I> could increase the NADH and ATP levels. </LI> <LI> The recombinant strains exhibited enhanced resistance to butyric acid and glucose. </LI> <LI> 48.2 g/L butyrate at a productivity of 0.50 g/L h and yield of 0.38 g/g was obtained. </LI> </UL> </P>