Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation.

Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions.

We 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 theL-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.

Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions.

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.

Increased fructose 1,6-bisphosphate aldolase in plastids enhances growth and photosynthesis of tobacco plants.

The Calvin cycle is the initial pathway of photosynthetic carbon fixation, and several of its reaction steps are suggested to exert rate-limiting influence on the growth of higher plants. Plastid fructose 1,6-bisphosphate aldolase (aldolase, EC 4.1.2.13) is one of the nonregulated enzymes comprising the Calvin cycle and is predicted to have the potential to control photosynthetic carbon flux through the cycle. In order to investigate the effect of overexpression of aldolase, this study generated transgenic tobacco (Nicotiana tabacum L. cv Xanthi) expressing Arabidopsis plastid aldolase. Resultant transgenic plants with 1.4-1.9-fold higher aldolase activities than those of wild-type plants showed enhanced growth, culminating in increased biomass, particularly under high CO2 concentration (700 ppm) where the increase reached 2.2-fold relative to wild-type plants. This increase was associated with a 1.5-fold elevation of photosynthetic CO2 fixation in the transgenic plants. The increased plastid aldolase resulted in a decrease in 3-phosphoglycerate and an increase in ribulose 1,5-bisphosphate and its immediate precursors in the Calvin cycle, but no significant changes in the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) or other major enzymes of carbon assimilation. Taken together, these results suggest that aldolase overexpression stimulates ribulose 1,5-bisphosphate regeneration and promotes CO2 fixation. It was concluded that increased photosynthetic rate was responsible for enhanced growth and biomass yields of aldolase-overexpressing plants.

Alteration of photosynthate partitioning by high-level expression of phosphoglucomutase in tobacco chloroplasts.

Plastidial phosphoglucomutase (PGM) plays an important role in starch synthesis and degradation. Nonetheless, the impact of enhanced plastidial PGM activity on metabolism in photosynthetic tissue is yet to be elucidated. In this study, we generated transplastomic tobacco plants overproducing Arabidopsis thaliana plastidial PGM (AtptPGM) in chloroplasts and analyzed the consequent metabolic and physiological parameters in the transplastomic plants. AtptPGM accumulated in the chloroplasts to up to 16% of total soluble protein in the leaves. PGM activity in leaves increased 100-fold relative to that of wild-type plants. The transplastomic plants were phenotypically indistinguishable in their growth rates, photosynthetic activities, and starch synthesis from wild-type plants, but hexose partitioning in the light period was dramatically different. Furthermore, alteration of extracellular invertase activity was observed in the lower leaves of the transplastomic plants. These observations suggest that high-level expression of plastidial PGM alters hexose partitioning in light periods via modification of extracellular invertase activity.

Role and regulation of cAMP in seed germination of Phacelia tanacetifolia.

Although adenosine 3',5'-cyclic monophosphate (cAMP) is known as a key second messenger in many living organisms, regulating a wide range of cellular responses, its biological function in higher plants is not well understood. In this study, the role and the regulation mechanism of cAMP in seed germination of Phacelia tanacetifolia Benth. were examined. The cAMP level of the seeds incubated under optimal conditions for germination showed a transient elevation before germination. When the seeds were exposed to light or supraoptimal temperature during incubation, elevation of cAMP levels as well as germination of the seeds were inhibited. Addition of membrane-permeable cAMP to the medium restored the germination rates of these seeds, suggesting that cAMP functions during germination. Treatment of the seeds with gibberellin (GA) was also effective to restore the elevation of cAMP levels and germination of the seeds. Uniconazole, a potent inhibitor of GA biosynthesis, blocked elevation of cAMP level under optimal conditions for germination. These results suggest that cAMP plays a role in the regulation of germination and that the cAMP level is regulated by GA in P. tanacetifolia seeds.

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis.

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.

SWAP-70 is important for invasive phenotypes of mouse embryo fibroblasts transformed by v-Src.

SWAP-70 is a protein involved in actin rearrangement, especially in membrane ruffling. Mouse embryo fibroblasts (MEFs) deficient in SWAP-70 show impaired membrane ruffling and fail to grow in soft agar after transformation by v-Src. Here, we show that v-Src transformed MEFs expressing SWAP-70 are highly invasive. MEFs expressing SWAP-70 or v-Src alone were far less invasive, suggesting that both proteins were required for the cells to be invasive. Expression of both SWAP-70 and v-Src induced constant membrane ruffling, which may cause vigorous cell movement, probably required for invasiveness of the cells. Expression of v-Src alone morphologically transformed MEFs but formed lamellipodia rather than membrane ruffles, suggesting less aggressive nature of the cells compared with those expressing both SWAP-70 and v-Src. These results suggest that v-Src and SWAP-70 act synergistically in the invasion activity of MEFs.