Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

Abstract

We previously reported that Corynebacterium glutamicum strain ⌬ldhA⌬ppc؉alaD؉gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159 -165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/ pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/ pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD ؉ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copynumber plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

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

“…To address the reduced glucose consumption under oxygen deprivation in the ⌬LDH⌬PEPC background, we chromosomally integrated and overexpressed glycolytic pathway genes gapA, pyk, pfkA, pgi, and tpi in accordance with our previous findings (20,27). These genes encode GAPDH, pyruvate kinase, phosphofructokinase, phosphoglucose isomerase, and triosephosphate isomerase, respectively ( Table 1).…”

“…Primers, plasmids, and strains used and constructed in this study are listed in Tables 1 and 2. Chromosomal gene deletion, insertion, and substitution were achieved via a markerless system using suicide vector pCRA725 carrying the sacB gene (19,27,28). Plasmids were transformed into C. glutamicum by electroporation (29).…”

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).…”