Improvement of the Redox Balance Increasesl-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

Abstract

Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD ؉ ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD ؉ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose ؊1 , and the L-valine productivity reached 1,940 mM after 48 h.

Uncovering rare NADH-preferring ketol-acid reductoisomerases

“…Based on previous structural studies and mutational analysis (Brinkmann-Chen et al, 2013;Hasegawa et al, 2012;Rane and Calvo, 1997), we have proposed that cofactor specificity is controlled by up to four amino acid residues in the loop: the first two positions, the antepenultimate position, and the ultimate position, as indicated by red arrows in Figure 1 and labeled for the S. exigua KARI in Figure 2. We searched the expanded sequence alignment for proteins having an acidic residue at any of these four positions.…”

“…It is interesting that the plurality of these putative NADH-utilizing KARIs have acidic residues the first position of the E2DB-loop. Although none of our previous cofactor-specificity reversals required an acidic residue at this position, Hasegawa et al introduced a mutation to glutamate at this position, suggested by comparison with the NADH-dependent dihydrolipoamide dehydrogenases, in an attempt to reverse the cofactor specificity of the C. glutamicum KARI (Hasegawa et al, 2012).…”

“…However, finding a sequence with specific desired properties can be difficult, particularly when only a few members of a protein family have been characterized and a detailed understanding of the structure-function relationship is lacking. The ketol-acid reductoisomerase (KARI, EC 1.1.1.86, also known as acetohydroxyacid isomeroreductase (AHAIR)) enzymes have attracted much interest for production of amino acids and biofuels (Atsumi et al, 2008a;Bastian et al, 2011;Brinkmann-Chen et al, 2013;Hasegawa et al, 2012;Liu et al, 2010). These oxidoreductases catalyze the second step in the branched chain amino-acid (BCAA) biosynthesis pathway (Chunduru et al, 1989), conversion of (S)-2-acetolactate (S2AL) to (R)-2,3-dihydroxyisovalerate (RDHIV) via a methyl shift coupled to a reduction with concomitant oxidation of a nicotinamide adenine dinucleotide cofactor.…”

Conserved Pyridoxal Protein That Regulates Ile and Val Metabolism

“…Activity of acetohydroxy acid synthase (AHAS) was determined in the reaction mixture containing 100 mM potassium phosphate buffer, pH 7.5, 10 mM MgCl 2 , 50 mM pyruvate, 100 M thiamine pyrophosphate (TPP), 100 M flavin adenine dinucleotide (FAD), and the cell-free lysate (ϳ300 g of protein) in a total volume of 500 l at 30°C. The rate of pyruvate consumption was calculated by monitoring the decrease of the absorbance at 333 nm (ε, 17.5 M cm Ϫ1 ) (12). (iii) Transaminase B (IlvE) activity.…”

Transformation of Biomass into Commodity Chemicals Using Enzymes or Cells

“…In this way a titer of 227 g/L has been achieved with a productivity of 4.7 g/(L h) with yields up to 0.41 g/g. 394 By further eliminating side-reactions, the yield has been increased to 0.57 g/g, at a titer of 150 g/L and a productivity 6.3 g/(L h). …”