Engineering of Corynebacterium glutamicum for High-Yieldl-Valine Production under Oxygen Deprivation Conditions

Abstract

bWe previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of L-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving the L-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD ؉ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose ؊1 after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Carbon Flux Analysis by 13 C Nuclear Magnetic Resonance To Determine the Effect of CO 2 on Anaerobic Succinate Production by Corynebacterium glutamicum

“…In dense cell suspensions (OD 600 ϭ 100), end products and glucose were determined online by in vivo NMR, while in diluted cell suspensions (OD 600 ϭ 7.5), they were measured by NMR and HPLC offline in the sample supernatants. The pH of the cell suspension was maintained at 7.0 or 5.7. b Product yields were calculated assuming that [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose was processed via the EMP pathway and that it produces a labeled and an unlabeled pyruvate molecule as follows: lactate yield ϭ [3-material). This trend was even more pronounced when the pure CO 2 was bubbled through the cell suspension (Fig.…”

“…Cells produced a small amount of [1,2-13 C]lactate (about 1% of total lactate) from the metabolism of [2][3][4][5][6][7][8][9][10][11][12][13] C]glucose, regardless of the CO 2 content of the atmosphere (Fig. 2D).…”

“…In contrast, in the absence of oxygen and nitrate, cell growth is negligible, while the ability to metabolize sugars is retained (5,6). Thus, cells of C. glutamicum can be grown aerobically and subsequently used for fermentation of desired products under anoxic conditions, as demonstrated for L-alanine, L-valine, or isobutanol (7)(8)(9)(10).…”

Characterization and Molecular Mechanism of AroP as an Aromatic Amino Acid and Histidine Transporter in Corynebacterium glutamicum

“…Corynebacterium glutamicum, a Gram-positive soil bacterium with high GϩC content, is widely used for large-scale production of amino acids and is considered to be an ideal model to study amino acid metabolism and transport (21,22). The genome of C. glutamicum carries approximately 750 membrane proteins that could possibly function as transporters, and members of 17 transporter families are predicted to be involved in amino acid transport (1).…”

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

“…This is the first confirmation of a bacterial arabino-xylooligosaccharide transporter, to the best of our knowledge. Given the efficacy of C. glutamicum to produce a variety of chemicals, including succinate (10), ethanol (11), isobutanol (12), alanine (13,14), and valine (15), this study could contribute to better understanding and optimization of utilization of the arabinoxylan component of lignocellulosic biomass by C. glutamicum and further enhance the utility of the microorganism in the production of more valuable products from lignocellulosic biomass.…”