Corynebacterium glutamicum tailored for efficient isobutanol production

Abstract

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 ilvBNCD 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 pntAB 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 adhA 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+ 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 Y(P/S) of about 0.48 mol per mol of glucose in the production phase.

Fig. 1.

Enzymes of the central metabolism with the biosynthetic pathway of l-valine in C. glutamicum and the synthetic pathway from ketoisovalerate to isobutanol. Abbreviations: Adh, alcohol dehydrogenase; AHAIR, acetohydroxyacid isomeroreductase; AHAS, acetohydroxyacid synthase; AK, acetate kinase; DHAD, dihydroxyacid dehydratase; FUM, fumarase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KIVD, 2-ketoacid decarboxylase from L. lactis; LdhA, l-lactate dehydrogenase; MalE, malic enzyme; Mdh, malate dehydrogenase; MQO, malate:quinone oxidoreductase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PEPCk, PEP carboxykinase; PEPCx, PEP carboxylase; PK, pyruvate kinase; PntAB, membrane bound transhydrogenase from E. coli; PTA, phosphotransacetylase; PQO, pyruvate:quinone oxidoreductase; SDH, succinate dehydrogenase; TA, transaminase B; TCA, tricarboxylic acid.

Fig. 2.

OD, glucose consumption, and l-lactate, succinate, and isobutanol formation of (A) C. glutamicum ΔaceE Δpqo ΔilvE(pJC4ilvBNCD) (C. glutamicum Iso1) and (B) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA(pJC4ilvBNCD)(pBB1kivd-adh2) (C. glutamicum Iso3) cultivated in Müller-Krempel bottles filled with CGXII medium containing about 100 mM glucose, 0.5% (wt/vol) yeast extract, and l-valine, l-isoleucine, and l-leucine (2 mM each). ▾, OD₆₀₀; ■, glucose; □, succinate; ♢, l-lactate; •, isobutanol. Three independent fermentations were performed. Error bars show standard deviations.

Fig. 3.

OD, glucose consumption, and l-lactate, succinate, and isobutanol formation of (A) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD)(pBB1kivd-adh2) (C. glutamicum Iso4) and (B) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adh2) (C. glutamicum Iso5) cultivated in Müller-Krempel bottles filled with CGXII medium containing about 100 mM glucose, 0.5% (wt/vol) yeast extract, and l-valine, l-isoleucine, and l-leucine (2 mM each). ▾, OD₆₀₀; ■, glucose; □, succinate; ♢, l-lactate; •, isobutanol. Three independent fermentations were performed. Error bars show standard deviations.

Fig. 4.

OD, glucose consumption, and l-lactate, succinate, and isobutanol formation of (A) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA(pJC4ilvBNCD)(pBB1kivd) (C. glutamicum Iso6) and (B) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA) (C. glutamicum Iso7) cultivated in Müller-Krempel bottles filled with CGXII medium containing about 100 mM glucose, 0.5% (wt/vol) yeast extract, and l-valine, l-isoleucine, and l-leucine (2 mM each). ▾, OD₆₀₀; ■, glucose; □, succinate; ♢, l-lactate; •, isobutanol. Three independent fermentations were performed. Error bars show standard deviations.

Fig. 5.

OD, glucose consumption, and l-lactate, succinate, and isobutanol formation of (A) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA ΔmalE(pJC4ilvBNCD)(pBB1kivd-adh2) (C. glutamicum Iso8) and (B) C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh ΔmalE(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA) (C. glutamicum Iso9) cultivated in Müller-Krempel bottles filled with CGXII medium containing about 100 mM glucose, 0.5% (wt/vol) yeast extract, and l-valine, l-isoleucine, and l-leucine (2 mM each). ▾, OD₆₀₀; ■, glucose; □, succinate; ♢, l-lactate; •, isobutanol. Three independent fermentations were performed. Error bars show standard deviations.

Fig. 6.

Isobutanol accumulation during a representative fed-batch fermentation of C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA) (C. glutamicum Iso7) on CGXII medium initially containing 4% (wt/vol) glucose, 1% (wt/vol) acetate, 0.5% (wt/vol) yeast extract, and 2 mM l-valine, l-isoleucine, and l-leucine, respectively. After 9.5 h, the aeration was switched off and the stirring speed was reduced to 300 rpm. ▾, OD₆₀₀; ■, glucose; ○, acetate; □, succinate; ♢, pyruvate; •, isobutanol. Three independent fed-batch fermentations were performed, all three showing comparable results.