Corynebacterium glutamicum Tailored for Efficient Isobutanol Production

We recently engineeredCorynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of theilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineeredC. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation ofl -lactate and malate dehydrogenases, implementation of ketoacid decarboxylase fromLactococcus lactis , alcohol dehydrogenase 2 (ADH2) fromSaccharomyces cerevisiae , and expression of thepntAB transhydrogenase genes fromEscherichia coli . The resulting strain produced isobutanol with a substrate-specific yield (YP/S ) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 fromS. cerevisiae was involved in isobutanol formation withC. glutamicum , and overexpression of the correspondingadhA gene increased the YP/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the YP/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+ to NADPH+H+ . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain,C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh (pJC4ilvBNCD-pntAB )(pBB1kivd-adhA ), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h−1 , and showed an overall YP/S of about 0.48 mol per mol of glucose in the production phase.