Inclusion of maintenance energy improves the intracellular flux predictions of CHO.

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.

Towards rational glyco-engineering in CHO: from data to predictive models.

Metabolic modelling strives to develop modelling approaches that are robust and highly predictive. To achieve this, various modelling designs, including hybrid models, and parameter estimation methods that define the type and number of parameters used in the model, are adapted. Accurate input data play an important role so that the selection of experimental methods that provide input data of the required precision with low measurement errors is crucial. For the biopharmaceutically relevant protein glycosylation, the most prominent available models are kinetic models which are able to capture the dynamic nature of protein N-glycosylation. In this review we focus on how to choose the most suitable model for a specific research question, as well as on parameters and considerations to take into account before planning relevant experiments.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

Error propagation in constraint-based modeling of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the most popular mammalian cell factories for the production of glycosylated biopharmaceuticals. To further increase titer and productivity and ensure product quality, rational system-level engineering strategies based on constraint-based metabolic modeling, such as flux balance analysis (FBA), have gained strong interest. However, the quality of FBA predictions depends on the accuracy of the experimental input data, especially on the exchange rates of extracellular metabolites. Yet, it is not standard practice to devote sufficient attention to the accurate determination of these rates. In this work, we investigated to what degree the sampling frequency during a batch culture and the measurement errors of metabolite concentrations influence the accuracy of the calculated exchange rates and further, how this error then propagates into FBA predictions of growth rates. We determined that accurate measurements of essential amino acids with low uptake rates are crucial for the accuracy of FBA predictions, followed by a sufficient number of analyzed time points. We observed that the measured difference in growth rates of two cell lines can only be reliably predicted when both high measurement accuracy and sampling frequency are ensured.

What CHO is made of: Variations in the biomass composition of Chinese hamster ovary cell lines.

BACKGROUND: Cell line-specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) cells. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions. RESULTS: We report total protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and detailed protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line- and condition-specific biomass functions for use in cell line-specific, genome-scale metabolic models of CHO. Finally, flux balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis. CONCLUSIONS: Our study reveals a strong variability of the total protein content and cell dry mass across cell lines. However, the relative amino acid composition is independent of the cell line and condition and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and thus will have to be determined in each case. These cell line-specific variations in biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are rather dominated by inaccuracies in the exchange rate spectra. Cell-specific biomass variations only become important if the experimental errors in the exchange rate spectra drop below twenty percent.

Robust Analytical Methods for the Accurate Quantification of the Total Biomass Composition of Mammalian Cells.

Biomass composition is an important input for genome-scale metabolic models and has a big impact on their predictive capabilities. However, researchers often rely on generic data for biomass composition, e.g. collected from similar organisms. This leads to inaccurate predictions, because biomass composition varies between different cell lines, conditions, and growth phases. In this chapter we present protocols for the determination of the biomass composition of Chinese Hamster Ovary (CHO) cells. These methods can easily be adapted to other types of mammalian cells. The protocols include the quantification of cell dry mass and of the main biomass components, namely protein, lipid, DNA, RNA, and carbohydrates. Cell dry mass is determined gravimetrically by weighing a defined number of cells. Amino acid composition and protein content are measured by gas chromatography mass spectrometry. Lipids are quantified by shotgun mass spectrometry, which provides quantities for the different lipid classes and also the distribution of fatty acids. RNA is purified and then quantified spectrophotometrically. The methods for DNA and carbohydrates are simple fluorometric and colorimetric assays adapted to a 96-well plate format. To ensure quantitative results, internal standards or spike-in controls are used in all methods, e.g. to account for possible matrix effects or loss of material. Finally, the last section provides a guide on how to convert the measured data into biomass equations, which can then be integrated into a metabolic model.

Genetic and Epigenetic Variation across Genes Involved in Energy Metabolism and Mitochondria of Chinese Hamster Ovary Cell Lines.

The increasingdemandfor biopharmaceutical products drives the search for efficient cell factories that are able to sustainably support rapid growth, high productivity, and product quality. As these depend on energy generation, here the genomic variation in nuclear genes associated with mitochondria and energy metabolism and the mitochondrial genome of 14 cell lines is investigated. The variants called enable reliable tracing of lineages. Unique sequence variations are observed in cell lines adapted to grow in protein-free media, enriched in signaling pathways or mitogen-activated protein kinase 3. High-producing cell lines bear unique mutations in nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND2 and ND4) and in peroxisomal acyl-CoA synthetase (ACSL4), involved in lipid metabolism. As phenotypes are determined not only by functional mutations, but also by the exquisite regulation of expression patterns, it is not surprising that ≈50% of the genes investigated here are found to be differentially methylated and thus epigenetically controlled, enabling a clear distinction of high producers, and cells adapted to a minimal, glutamine (Gln)-free medium. Similar pathways are enriched as those identified by genome variation. This strengthens the hypothesis that these phenomena act together to define cell behavior.

Designing Optimized Production Hosts by Metabolic Modeling.

Many of the complex and expensive production steps in the chemical industry are readily available in living cells. In order to overcome the metabolic limits of these cells, the optimal genetic intervention strategies can be computed by the use of metabolic modeling. Elementary flux mode analysis (EFMA) is an ideal tool for this task, as it does not require defining a cellular objective function. We present two EFMA-based methods to optimize production hosts: (1) the standard approach that can only be used for small and medium scale metabolic networks and (2) the advanced dual system approach that can be utilized to directly compute intervention strategies in a genome-scale metabolic model.

CHOmine: an integrated data warehouse for CHO systems biology and modeling.

The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. Database URL: http://www.chogenome.org.

What can mathematical modelling say about CHO metabolism and protein glycosylation?

Chinese hamster ovary cells have been in the spotlight for process optimization in recent years, due to being the major, long established cell factory for the production of recombinant proteins. A deep, quantitative understanding of CHO metabolism and mechanisms involved in protein glycosylation has proven to be attainable through the development of high throughput technologies. Here we review the most notable accomplishments in the field of modelling CHO metabolism and protein glycosylation.

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism.

Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Avoiding the Enumeration of Infeasible Elementary Flux Modes by Including Transcriptional Regulatory Rules in the Enumeration Process Saves Computational Costs.

Despite the significant progress made in recent years, the computation of the complete set of elementary flux modes of large or even genome-scale metabolic networks is still impossible. We introduce a novel approach to speed up the calculation of elementary flux modes by including transcriptional regulatory information into the analysis of metabolic networks. Taking into account gene regulation dramatically reduces the solution space and allows the presented algorithm to constantly eliminate biologically infeasible modes at an early stage of the computation procedure. Thereby, computational costs, such as runtime, memory usage, and disk space, are extremely reduced. Moreover, we show that the application of transcriptional rules identifies non-trivial system-wide effects on metabolism. Using the presented algorithm pushes the size of metabolic networks that can be studied by elementary flux modes to new and much higher limits without the loss of predictive quality. This makes unbiased, system-wide predictions in large scale metabolic networks possible without resorting to any optimization principle.

Predicting genetic engineering targets with Elementary Flux Mode Analysis: a review of four current methods.

Elementary flux modes (EFMs) are a well-established tool in metabolic modeling. EFMs are minimal, feasible, steady state pathways through a metabolic network. They are used in various approaches to predict targets for genetic interventions in order to increase production of a molecule of interest via a host cell. Here we give an introduction to the concept of EFMs, present an overview of four methods which use EFMs in order to predict engineering targets and lastly use a toy model and a small-scale metabolic model to demonstrate and compare the capabilities of these methods.

Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism.

Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

Metabolomics integrated elementary flux mode analysis in large metabolic networks.

Elementary flux modes (EFMs) are non-decomposable steady-state pathways in metabolic networks. They characterize phenotypes, quantify robustness or identify engineering targets. An EFM analysis (EFMA) is currently restricted to medium-scale models, as the number of EFMs explodes with the network's size. However, many topologically feasible EFMs are biologically irrelevant. We present thermodynamic EFMA (tEFMA), which calculates only the small(er) subset of thermodynamically feasible EFMs. We integrate network embedded thermodynamics into EFMA and show that we can use the metabolome to identify and remove thermodynamically infeasible EFMs during an EFMA without losing biologically relevant EFMs. Calculating only the thermodynamically feasible EFMs strongly reduces memory consumption and program runtime, allowing the analysis of larger networks. We apply tEFMA to study the central carbon metabolism of E. coli and find that up to 80% of its EFMs are thermodynamically infeasible. Moreover, we identify glutamate dehydrogenase as a bottleneck, when E. coli is grown on glucose and explain its inactivity as a consequence of network embedded thermodynamics. We implemented tEFMA as a Java package which is available for download at https://github.com/mpgerstl/tEFMA.

Benzo annulated cycloheptatriene PCP pincer iridium complexes.

The benzo annulated cycloheptatriene PCP pincer ligand was prepared in five steps. Treatment of with Ir(CO)3Cl gave the meridional cyclometalated chlorohydrido carbonyl iridium complexes which differ in their arrangement of the H, Cl, and CO ligands around iridium. Storing in THF led to isomerization processes. Hydrogen shifts from the sp(3)-CH carbon bound to iridium into the ligand backbone produced the three isomers . Reductive elimination of HCl from these complexes resulted in the square planar Ir(i) carbonyl complexes . Abstraction of the hydrogen from the sp(3)-CH-Ir fragment could be achieved either by treatment of with Ph3CBF4 or by the elimination of H2 which is initiated by CF3SO3H. The mass spectrometric characterisation of using fast atom bombardment reveals a complex fragmentation pattern. These different "fragment" ions were further investigated by electro-spray ionisation (tandem) mass spectrometry in high and low resolution. The identified compounds were attributed to structures by DFT calculations.

Design of optimally constructed metabolic networks of minimal functionality.

BACKGROUND: Metabolic engineering aims to design microorganisms that will generate a product of interest at high yield. Thus, a variety of in silico modeling strategies has been applied successfully, including the concepts of elementary flux modes (EFMs) and constrained minimal cut sets (cMCSs). The EFMs (minimal, steady state pathways through the system) can be calculated given a metabolic model. cMCSs are sets of reaction deletions in such a network that will allow desired pathways to survive and disable undesired ones (e.g., those with low product secretion or low growth rates). Grouping the modes into desired and undesired categories had to be done manually until now. RESULTS: Although the optimal solution for a given set of pathways will always be found with the currently available tools, manual selection may lead to a sub-optimal solution with respect to a metabolic engineering target. A small change in the selection of modes can reduce the number of necessary deletions while only slightly reducing production. Based on our recently introduced formulation of cut set calculations using binary linear programming, we suggest an algorithm that does not require manual selection of the desired pathways. CONCLUSIONS: We demonstrated the principle of our algorithm with the help of a small toy network and applied it to a model of E. coli using different design objectives. Furthermore we validated our method by reproducing previously obtained results without requiring manual grouping of modes.

Elementary flux modes in a nutshell: properties, calculation and applications.

Elementary flux mode (EFM) analysis allows the unbiased decomposition of a metabolic network into minimal functional units, making it a powerful tool for metabolic engineering. While the use of EFM analysis (EFMA) is still limited by the size of the models it can handle, EFMA has been successfully applied to solve real-world metabolic engineering problems. Here we provide a user-oriented introduction to EFMA, provide examples of recent applications, analyze current research strategies to overcome the computational restrictions and give an overview over current approaches, which aim to identify and calculate only biologically relevant EFMs.

regEfmtool: speeding up elementary flux mode calculation using transcriptional regulatory rules in the form of three-state logic.

Despite the considerable progress made in recent years, the computation of the complete set of elementary flux modes of large or even genome-scale metabolic networks is still impossible. We present regEfmtool which is an extension to efmtool that utilizes transcriptional regulatory networks for the computation of elementary flux modes. The implemented extension significantly decreases the computational costs for the calculation of elementary flux modes, such as runtime, memory usage and disk space by omitting biologically infeasible solutions. Hence, using the presented regEfmtool pushes the size of metabolic networks that can be studied by elementary flux modes to new limits.

Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth.

Among the vast variety of Saccharomyces cerevisiae strains, the BY family is particularly important because the widely used deletion collections are based on this background. Here we demonstrate that some standard growth media recipes require substantial modifications to provide optimum growth conditions for auxotrophic BY strains and to avoid growth arrest before glucose is depleted. In addition to the essential supplements that are required to satisfy auxotrophic requirements, we found the four amino acids phenylalanine, glutamic acid, serine, and threonine to be indispensable for optimum growth, despite the fact that BY is 'prototrophic' for these amino acids. Interestingly, other widely used S. cerevisiae strains, such as strains of the CEN.PK family, are less sensitive to lack of the described supplements. Furthermore, we found that the concentration of inositol in yeast nitrogen base is too low to support fast proliferation of yeast cultures until glucose is exhausted. Depletion of inositol during exponential growth induces characteristic changes, namely a decrease in glucose uptake and maximum specific growth rate, increased cell size, reduced viability, and accumulation of lipid storage pools. Thus, several of the existing growth media recipes need to be revised to achieve optimum growth conditions for BY-derived strains.