Overexpression of peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺) in Chinese hamster ovary cells increases oxidative metabolism and IgG productivity.

Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

Systematic identification and elimination of flux bottlenecks in the aldehyde production pathway of Synechococcus elongatus PCC 7942.

Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.

Glutamate-oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux.

Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the endoplasmic reticulum to mitochondria of palmitate-treated hepatocytes activates anaplerotic flux from glutamine to α-ketoglutarate (αKG), which subsequently enters the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fuels elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared with media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase activity significantly reduced the lipotoxic effects of palmitate, whereas knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. 13C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.

An integrated network analysis identifies how ArcAB enables metabolic oscillations in the nitric oxide detoxification network of Escherichia coli.

The virulences of many pathogens depend on their abilities to detoxify the immune antimicrobial nitric oxide (NO•). The functions of bacterial NO• detoxification machinery depend on oxygen (O2 ), with O2 inhibiting some enzymes, whereas others use it as a substrate. Previously, Escherichia coli NO• detoxification was found to be highly attenuated under microaerobic conditions and metabolic oscillations were observed. The oscillations in [NO•] and [O2 ] were found to result from the inhibitory action of NO• on aerobic respiration, the catalytic inactivation of NO• by Hmp (an NO• dioxygenase), and an imbalanced competition for O2 between Hmp and cytochrome terminal oxidase activity. Here the authors investigated the role of the ArcAB two component system (TCS) in microaerobic NO• detoxification. The authors observed that wild-type, ΔarcA, and ΔarcB had comparable initial NO• clearance times; however, the mutant cultures failed to exhibit [NO•] and [O2 ] oscillations. Using an approach that employed experimentation and computational modeling, the authors found that the loss of oscillations in ΔarcA was due to insufficient induction of cytochrome bd-I expression. Collectively, these results establish ArcAB as a TCS that influences NO• detoxification in E. coli within the physiologically-relevant microaerobic regime.

The precipitation of indium at elevated pH in a stream influenced by acid mine drainage.

Indium is an increasingly important metal in semiconductors and electronics and has uses in important energy technologies such as photovoltaic cells and light-emitting diodes (LEDs). One significant flux of indium to the environment is from lead, zinc, copper, and tin mining and smelting, but little is known about its aqueous behavior after it is mobilized. In this study, we use Mineral Creek, a headwater stream in southwestern Colorado severely affected by heavy metal contamination as a result of acid mine drainage, as a natural laboratory to study the aqueous behavior of indium. At the existing pH of ~3, indium concentrations are 6-29µg/L (10,000× those found in natural rivers), and are completely filterable through a 0.45µm filter. During a pH modification experiment, the pH of the system was raised to >8, and >99% of the indium became associated with the suspended solid phase (i.e. does not pass through a 0.45µm filter). To determine the mechanism of removal of indium from the filterable and likely primarily dissolved phase, we conducted laboratory experiments to determine an upper bound for a sorption constant to iron oxides, and used this, along with other published thermodynamic constants, to model the partitioning of indium in Mineral Creek. Modeling results suggest that the removal of indium from the filterable phase is consistent with precipitation of indium hydroxide from a dissolved phase. This work demonstrates that nonferrous mining processes can be a significant source of indium to the environment, and provides critical information about the aqueous behavior of indium.