Kinetic model optimization and its application to mitigating the Warburg effect through multiple enzyme alterations

O'Brien, Conor M.; Allman, Andrew; Daoutidis, Pródromos; Hu, Wei Shou

doi:10.1016/j.ymben.2019.08.005

Cited by 8 publications

(7 citation statements)

References 49 publications

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“…Furthermore, several other byproducts of the amino acid metabolism (e.g., formate, homocysteine or indolelactate) as well as metabolites originating from lipid metabolism were determined to be detrimental to CHO growth [173,174]. To overcome these issues, numerous successful interventions, such as engineering amino acid catabolism, were performed to reduce the accumulation of toxic metabolites and to improve growth and product titers of CHO cell cultures [157,[175][176][177].…”

Section: Making Metabolism More Efficientmentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.

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Section: Making Metabolism More Efficientmentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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“…Mathematical modelling of Warburg metabolism has already been addressed in several studies, mostly for the purpose of a deeper understanding of tumour metabolism [ 32 , 33 , 34 , 35 ] or pathway engineering [ 36 ]. However, they mostly focus on specific participants in the metabolic pathways rather than the regulatory network of metabolism.…”

Section: Introductionmentioning

confidence: 99%

Therapeutic Approach of KRAS Mutant Tumours by the Combination of Pharmacologic Ascorbate and Chloroquine

2021

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The Warburg effect has been considered a potential therapeutic target to fight against cancer progression. In KRAS mutant cells, PKM2 (pyruvate kinase isozyme M2) is hyper-activated, and it induces GLUT1 expression; therefore, KRAS has been closely involved in the initiation of Warburg metabolism. Although mTOR (mammalian target of rapamycin), a well-known inhibitor of autophagy-dependent survival in physiological conditions, is also activated in KRAS mutants, many recent studies have revealed that autophagy becomes hyper-active in KRAS mutant cancer cells. In the present study, a mathematical model was built containing the main elements of the regulatory network in KRAS mutant cancer cells to explore the further possible therapeutic strategies. Our dynamical analysis suggests that the downregulation of KRAS, mTOR and autophagy are crucial in anti-cancer therapy. PKM2 has been assumed to be the key switch in the stress response mechanism. We predicted that the addition of both pharmacologic ascorbate and chloroquine is able to block both KRAS and mTOR pathways: in this case, no GLUT1 expression is observed, meanwhile autophagy, essential for KRAS mutant cancer cells, is blocked. Corresponding to our system biological analysis, this combined pharmacologic ascorbate and chloroquine treatment in KRAS mutant cancers might be a therapeutic approach in anti-cancer therapies.

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“…These efforts have not been able to eliminate the Warburg effect and thus result in fast growth without aerobic glycolysis 10 . O'Brien and coworkers proposed that the large provision of energy and key precursors for fast biomass synthesis requires a new homeostatic state which can only be achieved by alteration of multiple enzymes involved with central carbon metabolism ‐ including those associated with the pentose phosphate pathway, glycolysis, and tricarboxylic acid (TCA) cycle pathway 12 . The pH, NAD + /NADH ratio, pyruvate availability, and mitochondrial functions during lactate consumption are other venues for the driving forces behind CHO cells metabolically switching from aerobic glycolysis to a highly oxidative metabolism 13 .…”

Section: Introductionmentioning

confidence: 99%

“…The consumption of glutamine, glutamic acid, and aspartic acid was also discovered to influence the onset of the oxidative phosphorylation (OXPHOS) 14 . Furthermore, both flux balance analysis (FBA) 10,15 and kinetic model optimization, 12 have demonstrated that the simultaneous activation of the malate ‐ aspartate shuttle (MAS) and triose dihydroxyacetone‐phosphate (DHAP) synthesis can be alternative routes for regulating the NAD + /NADH ratio in the cytosol ‐ due to this activation leading to a highly reduced pool of nicotinamides created by steady glycolytic activity and concomitant lactate consumption. The strategy employed was to identify optimal solutions which would force the cells toward a low lactate production state via multiple enzyme activity modifications.…”

Section: Introductionmentioning

confidence: 99%

“…By predicting the upregulation of lactate dehydrogenase, pyruvate dehydrogenase, and downregulation of malic enzyme 1, the resulting cytosolic pool of pyruvate would decrease, and thus lower the driving force for lactate production. At the same time, it was predicted that upregulating pyruvate dehydrogenase and acetyl‐CoA synthetase would lead to optimization of the TCA cycle 12 …”

Section: Introductionmentioning

confidence: 99%

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Exometabolome profiling reveals activation of the carnitine buffering pathway in fed‐batch cultures ofCHOcells co‐fed with glucose and lactic acid

Vappiani

Eyster

Orzechowski

et al. 2021

Biotechnology Progress

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Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy‐based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi‐quantitative and time‐dependent analysis of the exometabolome. This study focused on the CHO cell's metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon‐buffering pathway. CHO cells exhibited a balance between intermediates from (i) amino acid catabolism, (ii) fatty acid β‐oxidation, and (iii) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3‐hydroxy‐methyl‐glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl‐CoA groups as well as an ability to maintain the acyl‐CoA: free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine‐buffering system, HMG, and mevalonate pathways.

show abstract

Kinetic model optimization and its application to mitigating the Warburg effect through multiple enzyme alterations

Cited by 8 publications

References 49 publications

Key Challenges in Designing CHO Chassis Platforms

Key Challenges in Designing CHO Chassis Platforms

Therapeutic Approach of KRAS Mutant Tumours by the Combination of Pharmacologic Ascorbate and Chloroquine

Exometabolome profiling reveals activation of the carnitine buffering pathway in fed‐batch cultures ofCHOcells co‐fed with glucose and lactic acid

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