Elucidating the role of copper in CHO cell energy metabolism using 13C metabolic flux analysis

¹³C‐metabolic flux analysis was used to understand copper deficiency‐related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein‐producing CHO cells. Stationary‐phase labeling experiments with U‐¹³C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed‐batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC‐MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%–79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%–23% and 74%, respectively) compared with the Cu‐containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper‐deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1179–1186, 2015     

The combined effect of constant water deficit and nitrogen supply on WUE, NUE and Δ13C in durum wheat potted plants

Water scarcity and nitrogen shortage are the main constraints on durum wheat productivity. This paper examines the combined effects of a constant water deficit and nitrogen supply (NS) on growth, photosynthesis, stomatal conductance (g_s) and transpiration, instantaneous and time‐integrated water use efficiency (WUE) and nitrogen use efficiency (NUE) and carbon isotope discrimination (Δ¹³C) in durum wheat genotypes grown in pots under greenhouse conditions. Three water levels (40%, 70% and 100% container capacity), two nitrogen doses (high and low N) and four genotypes were assayed in a total of 24 experimental treatments. Water and nitrogen treatments were imposed 2 weeks after plant emergence. The growth, nitrogen content and Δ¹³C of the shoot and the gas exchange in the flag leaf were determined about 2 weeks after anthesis. As expected, both water and NS had a strong positive effect on growth. However, a reduction in water supply had low effect decreasing photosynthesis and transpiration, Δ¹³C and NUE and increasing WUE. On the contrary, increasing the level of nitrogen supplied had a significant negative effect on g_s, which decreased significantly the ratio of intercellular to ambient CO₂ concentrations and Δ¹³C, and increased both instantaneous and time‐integrated WUE. In addition, a higher N level also negatively affected the instantaneous and time‐integrated NUE. The Δ¹³C of shoots correlated significantly and negatively with either instantaneous or time‐integrated measurements of WUE. Moreover, within each NS, Δ¹³C also correlated negatively with the integrated NUE. We concluded that under our experimental conditions, Δ¹³C gives information about the efficiency with which not just water but also nitrogen are used by the plant. In addition, this study illustrates that a steady water limitation may strongly affect biomass without consistent changes in WUE. The lack of effect of the different water regimes on gas exchange, WUE and Δ¹³C illustrate the importance of how stress is imposed during growth.     

Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: flux variability analysis in relation to ¹³C metabolic flux analysis

Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large‐scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior ¹³C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH₄⁺ assimilation. Analysis of the light‐dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO₂ efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the ¹³C flux studies, ribulose‐1,5‐bisphosphate carboxylase activity is predicted to account fully for the light‐dependent changes in carbon balance.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.     

Partial nitrate nutrition amends photosynthetic characteristics in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. <Emphasis Type="Italic">japonica</Emphasis>) differing in nitrogen use efficiency

Partial nitrate nutrition (PNN) was found to improve rice (Oryza sativa L. var. japonica) growth. However, how PNN is related to photosynthesis in rice cultivars with different nitrogen use efficiency (NUE) is still not clear. Two rice cultivars, Nanguang (high NUE) and Elio (low NUE), were grown under sole NH₄ ⁺ and PNN at a total nitrogen concentration of 2.86 mM. The dry weight, leaf area, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and gas exchange parameters were measured. Nitrogen and Rubisco contents in the newly expanded leaves of cv. Nanguang were similar to those of cv. Elio when only NH₄ ⁺ was supplemented in the nutrient solution. However, in cv. Nanguang, nitrogen and Rubisco contents increased under PNN than under sole NH₄ ⁺ nutrition. Higher nitrogen and Rubisco contents were recorded in cv. Nanguang than in cv. Elio under PNN. The ratio of carboxylation efficiency (CE) to Rubisco content in cv. Nanguang was 11 and 14% higher than that in cv. Elio under NH₄ ⁺ and PNN, respectively. CE was 14% higher in cv. Nanguang than that in cv. Elio. The results suggest that PNN causes an increase in photosynthesis in cv. Nanguang. It is concluded that differences in Rubisco activity, rather than stomatal limitation, are responsible for the differences in photosynthesis between the two cultivars. The presence of nitrate increases Rubisco content in rice with a high NUE, which leads to faster biomass accumulation at later growth stages.     

Relative contribution of environmental and nutritional variables to net primary production of Cynodon dactylon (Linn.) Pers in the riparian zone of a Three Gorges tributary

Our knowledge of fundamental drivers of terrestrial net primary production (NPP) is crucial for improving the predictability of ecosystem stability under global climate change. However, the patterns and determinants of NPP are not fully understood, especially in the riparian zone ecosystem disturbed by periodic drought–rewetting (DRW) cycles. The environmental (flooding time, pH, moisture, and clay content) and nutritional properties (soil organic carbon, total nitrogen, total phosphorus, ammonium (NH₄⁺‐N), nitrate (NO₃^‐‐N), and C:N:P stoichiometry) were investigated in the riparian zone of Pengxi River‐a typical tributary of Three Gorges Reservoir (TGR). Structure equation modeling was performed to evaluate the relative importance of environmental and nutritional properties on NPP of Cynodon dactylon (Linn.) Pers (C. dactylon)‐a dominating plant in the riparian zone of TGR. Our results indicated that NPP was much lower under much severe flooding stress. All of these variables could predict 46% of the NPP variance. Nutrient use efficiency (NUE) was one of the most critical predictor shaping the change of NPP. Specifically, flooding stress as a major driver had a direct positive effect on soil moisture and soil clay content. The soil clay content positively affects the soil C: N ratio, which further had an indirect negative impact on NPP by mediating NUE. Overall, our study provided a comprehensive analysis of the effects of the combined effect of environmental and nutrient factors on NPP and showed that continuous DRW cycles induced by hydrological regime stimulate the decrease of NPP of C. dactylon by changing NUE strategies. Further research is needed to explore the responses of NPP and NUE under different land use to DRW cycles and to investigate the DRW effects on the combined effect of environmental and nutrient factors by in situ experiments and long‐term studies.     

Carbon partitioning to mobile and structural fractions in poplar wood under elevated CO2 (EUROFACE) and N fertilization

To determine whether globally increasing atmospheric carbon dioxide (CO₂) concentrations can affect carbon partitioning between nonstructural and structural carbon pools in agroforestry plantations, Populus nigra was grown in ambient air (about 370 μmol mol^?1 CO₂) and in air with elevated CO₂ concentrations (about 550 μmol mol^?1 CO₂) using free‐air CO₂ enrichment (FACE) technology. FACE was maintained for 5 years. After three growing seasons, the plantation was coppiced and one half of each experimental plot was fertilized with nitrogen. Carbon concentrations and stocks were measured in secondary sprouts in seasons of active growth and dormancy during 2 years after coppicing. Although FACE, N fertilization and season had significant tissue‐specific effects on carbon partitioning to the fractions of structural carbon, soluble sugars and starch as well as to residual soluble carbon, the overall magnitude of these shifts was small. The major effect of FACE and N fertilization was on cell wall biomass production, resulting in about 30% increased above ground stocks of both mobile and immobile carbon pools compared with fertilized trees under ambient CO₂. Relative C partitioning between mobile and immobile C pools was not significantly affected by FACE or N fertilization. These data demonstrate high metabolic flexibility of P. nigra to maintain C‐homeostasis under changing environmental conditions and illustrate that nonstructural carbon compounds can be utilized more rapidly for structural growth under elevated atmospheric [CO₂] in fertilized agroforestry systems. Thus, structural biomass production on abandoned agricultural land may contribute to achieving the goals of the Kyoto protocol.     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

iTRAQ‐based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies

Male and female poplars (Populus cathayana Rehd.) respond differently to nitrogen (N) and phosphorus (P) deficiencies. In this study, an iTRAQ‐based quantitative proteomic analysis was performed. N and P deficiencies caused 189 and 144 proteins to change in abundance in males and 244 and 464 in females, respectively. Compared to N‐ and P‐deficient males, both N‐ and P‐deficient females showed a wider range of changes in proteins that are involved in amino acid, carbohydrate and protein metabolism, and the sexual differences were significant. When comparing the effects of N‐ and P‐deficiencies, N‐deficient females expressed more changes in proteins that are involved in stress responses and gene expression regulation, while P‐deficient females showed more changes in proteins that are involved in energy and lipid metabolism, stress responses and gene expression regulation. The quantitative RT‐PCR analysis of stress‐related proteins showed that males have a better expression correlation between mRNA and protein levels than do females. This study shows that P. cathayana females are more sensitive and have more rapid metabolic mechanisms when responding to N and P deficiencies than do males, and P deficiency has a wider range of effects on females than does N deficiency.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

Halving sunlight reveals no carbon limitation of aboveground biomass production in alpine grassland

In temperate alpine environments, the short growing season, low temperature and a slow nutrient cycle may restrict plant growth more than carbon (C) assimilation does. To test whether C is a limiting resource, we applied a shade gradient from ambient light to 44% (maximum shade) of incident photon flux density (PFD) in late successional, Carex curvula‐dominated alpine grassland at 2,580 m elevation in the Swiss central Alps for 3 years (2014–2016). Total aboveground biomass did not significantly decrease under reduced PFD, with a confidence interval ranging from +4% to ?15% biomass in maximum shade. Belowground biomass, of which more than 80% were fine roots, was significantly reduced by a mean of 17.9 ± 4.6% (±SE), corresponding to 228 g/m², in maximum shade in 2015 and 2016. This suggests reduced investments into water and nutrient acquisition according to the functional equilibrium concept. Specific leaf area (SLA) and maximum leaf length of the most abundant species increased with decreasing PFD. Foliar concentration of nonstructural carbohydrates (NSC) was reduced by 12.5 ± 4.3% under maximum shade (mean of eight tested species), while NSC concentration of belowground storage organs were unchanged in the four most abundant forbs. Furthermore, maximum shade lowered foliar δ¹³C by 1.56 ± 0.35‰ and increased foliar nitrogen concentrations per unit dry mass by 18.8 ± 4.1% across six species in 2015. However, based on unit leaf area, N concentrations were lower in shade (effect of higher SLA). Thus, while we found typical morphological and physiological plant responses to lower light, shading did not considerably affect seasonal aboveground biomass production of this alpine plant community within a broad range of PFD. This suggests that C is not a growth‐limiting resource, matching the unresponsiveness to in situ CO₂ enrichment previously reported for this type of grassland.     

Gross primary production responses to warming,elevated CO2, and irrigation: quantifying the drivers of ecosystem physiology in a semiarid grassland

Determining whether the terrestrial biosphere will be a source or sink of carbon (C) under a future climate of elevated CO₂ (eCO₂) and warming requires accurate quantification of gross primary production (GPP), the largest flux of C in the global C cycle. We evaluated 6 years (2007–2012) of flux‐derived GPP data from the Prairie Heating and CO₂ Enrichment (PHACE) experiment, situated in a grassland in Wyoming, USA. The GPP data were used to calibrate a light response model whose basic formulation has been successfully used in a variety of ecosystems. The model was extended by modeling maximum photosynthetic rate (A_max) and light‐use efficiency (Q) as functions of soil water, air temperature, vapor pressure deficit, vegetation greenness, and nitrogen at current and antecedent (past) timescales. The model fits the observed GPP well (R² = 0.79), which was confirmed by other model performance checks that compared different variants of the model (e.g. with and without antecedent effects). Stimulation of cumulative 6‐year GPP by warming (29%, P = 0.02) and eCO₂ (26%, P = 0.07) was primarily driven by enhanced C uptake during spring (129%, P = 0.001) and fall (124%, P = 0.001), respectively, which was consistent across years. Antecedent air temperature (Tair_ant) and vapor pressure deficit (VPD_ant) effects on A_max (over the past 3–4 days and 1–3 days, respectively) were the most significant predictors of temporal variability in GPP among most treatments. The importance of VPD_ant suggests that atmospheric drought is important for predicting GPP under current and future climate; we highlight the need for experimental studies to identify the mechanisms underlying such antecedent effects. Finally, posterior estimates of cumulative GPP under control and eCO₂ treatments were tested as a benchmark against 12 terrestrial biosphere models (TBMs). The narrow uncertainties of these data‐driven GPP estimates suggest that they could be useful semi‐independent data streams for validating TBMs.     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

The role of the chaetognath Sagitta gazellae in the vertical carbon flux of the Southern Ocean

Chaetognaths are among the most abundant predators in the Southern Ocean and are potentially important components in the biological carbon pump due to the production of large, fast-sinking fecal pellets. In situ S. gazellae abundance, fecal pellet production, sinking rates, carbon content, and vertical carbon fluxes were measured at the Lazarev Sea between December 2005 and January 2006. Sagitta gazellae produce fecal pellets that sink at speeds of 33–600 m day⁻¹ and have carbon contents of 0.01–0.8 mg C pellet⁻¹. Vertical carbon flux was later compared with the total carbon flux measured at 360 m depth at the study area. Rough estimates using published seasonal abundance of S. gazellae indicate that, at 360 m depth in the Lazarev Sea, this specie may contribute 12 and 5% of the total vertical carbon flux in winter (ice-covered) and summer (ice-free), respectively. Thus, the role of chaetognaths in the downward transport of organic matter may be far more important than previously thought.