PMC

2-Ketoisovalerate, l-valine, and l-isoleucine synthesis from two molecules of pyruvate or from pyruvate plus 2-ketobutyrate, respectively, in C. glutamicum. Abbreviations: AHAS, acetohydroxyacid synthase; AHAIR, acetohydroxyacid isomeroreductase; DHAD, dihydoxyacid dehydratase; TA, transaminase B.

Growth of C. glutamicum ΔilvE (circles) and C. glutamicum ΔaceE ΔilvE (triangles) in shake flasks in minimal medium with l-isoleucine, l-leucine, and l-valine (1 mM each) and 2% (wt/vol) glucose (open symbols) or 1% (wt/vol) glucose plus 1% (wt/vol) acetate (closed symbols). The figure shows a representative data set of three independent cultivations, all three showing comparable results.

Exemplary fed-batch fermentations of C. glutamicum ΔaceE ΔilvE (pJC4ilvBNCD) (A) and C. glutamicum ΔaceE Δpqo ΔilvE (pJC4ilvBNCD) (B) in minimal medium with glucose (•), acetate (▾), and l-isoleucine, l-leucine, and l-valine (1 mM each). Other symbols: ▴, growth; □, 2-ketoisovalerate. By-products: ⋄, l-alanine; ▵, l-valine; ○, pyruvate. The figure shows one representative data set of three independent experiments.

Growth (▴) and 2-ketoisovalerate production (□) of C. glutamicum ΔilvE (A), C. glutamicum ΔaceE ΔilvE (B), and C. glutamicum ΔaceE ΔilvE (pJC4ilvBNCD) (C) in shake flask experiments in minimal medium with glucose (•), acetate (▾), and l-isoleucine, l-leucine, and l-valine (1 mM each). By-products: ⋄, l-alanine; ▵, l-valine; ○, pyruvate. The figure shows one representative data set of three independent experiments, all three showing comparable results.

Comparison of final 2-ketoisovalerate (KIV) concentrations (A), substrate-specific product yields (Y_P/S in mol KIV per mol of glucose, given for the production phase t = 16 to 56 h) (B), and volumetric productivity (in mM KIV per h, given for the production phase t = 16 to 56 h) (C) in cultures of C. glutamicum ΔaceE ΔilvE (pJC4ilvBNCD) (□) and C. glutamicum ΔaceE Δpqo ΔilvE (pJC4ilvBNCD) (░⃞). Means are from three independent experiments; error bars indicate standard deviations.

Pyruvate-dependent specific activities of cell extracts of C. glutamicum WT AHAS in the presence (•) or absence (▴) of 2-ketoisovalerate (50 mM). The cells were grown in minimal medium containing 2% (wt/vol) of glucose and harvested at an OD₆₀₀ of ∼5. The inset shows a Hanes-Wilkinson plot of the same data used for the determination of K_m and V_max.