Improvement of the redox balance increases L-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.

Fig 1

Metabolic pathway of C. glutamicum under oxygen deprivation conditions (bold arrows, predominant flow) and the biosynthetic pathway of l-valine. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; OD, oxaloacetate decarboxylase; MDH, malate dehydrogenase; SDH, succinate dehydrogenase; MQ, menaquinone; NDH, NADH dehydrogenase; LDH, lactate dehydrogenase; AHAS, acetohydroxy acid synthase; AHAIR, acetohydroxy acid isomeroreductase (AHAIR*, NAD-preferring mutant); DHAD, dihydroxy acid dehydratase; TA, transaminase B; LeuDH, leucine dehydrogenase (L. sphaericus).

Fig 2

Amino acid sequence alignment of nicotinamide coenzyme-binding enzymes around the β-α-β motif in the Rossmann fold domain and the design of NAD-preferring mutant AHAIR of C. glutamicum. Conserved amino acid residues specific for NAD or NADP binding sites are indicated as follows: ●, the fingerprint regions of the β-α-β-fold (GXGXXGXXXG, NAD specific; GXGXXAXXXA, NADP specific); −, negatively charged amino acid residues (NAD specific); +, positively charged amino acid residues (NADP specific). Substituted amino acid residues (S34G, L48E, and R49F) of the mutant AHAIR of C. glutamicum are shaded. Swiss-Prot accession codes for dihydrolipoamide dehydrogenase: Saccharomyces cerevisiae, P09624; E. coli, P0A9P0; Pseudomonas fluorescens, P14218; C. glutamicum, A4QB06. Swiss-Prot accession codes for glutathione reductase: S. cerevisiae, P41921; E. coli, P06715; Pseudomonas aeruginosa, P23189. Swiss-Prot accession codes for acetohydroxy acid isomeroreductase: E. coli, P05793; Buchnera aphidicola, Q9RQ51; P. aeruginosa, Q9HVA2; C. glutamicum, A4QDN4. ¹⁾, reference ; cylinders, α-helixes; arrows, β-strands.

Fig 3

Relative activity of AHAIR using NADH as a cofactor in the presence of NADP⁺. ●, wild type; □, mutant. Substrate concentrations were 10 mM 2-acetolactate, 0.2 mM NADH, and variable amounts of NADP⁺. Specific activities of the wild-type and mutant AHAIRs in this reaction (without NADP⁺) were 0.71 U/mg and 0.45 U/mg, respectively. The data are averages from triplicate experiments.

Fig 4

l-Valine production by BN^GEC™DLD/ΔLDH under oxygen deprivation conditions. ■, l-valine; ♦, succinate; ▲, acetate; ●, alanine; □, glucose. l-Valine production, by-product formation, and glucose concentration were corrected for dilution caused by the addition of NH₃ solution and glucose throughout the reaction. The data are averages from three independent experiments.