초록
<P><B>ABSTRACT</B><P> We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the <I>ilvBNCD</I> genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l -lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis , alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae , and expression of the <I>pntAB</I> transhydrogenase genes from Escherichia coli . The resulting strain produced isobutanol with a substrate-specific yield (Y P/S ) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum , and overexpression of the corresponding <I>adhA</I> gene increased the Y P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y P/S , indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H <SUP>+</SUP> to NADPH+H <SUP>+</SUP> . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum Δ <I>aceE</I> Δ <I>pqo</I> Δ <I>ilvE</I> Δ <I>ldhA</I> Δ <I>mdh</I> (pJC4 <I>ilvBNCD-pntAB</I> )(pBB1 <I>kivd-adhA</I> ), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h <SUP>−1</SUP> , and showed an overall Y P/S of about 0.48 mol per mol of glucose in the production phase. </P></P>