Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells

Yip, Shirley; Ming, Zhou; Joly, John C.; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

doi:10.1007/s12033-014-9762-0

Cited by 16 publications

(11 citation statements)

References 22 publications

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“…Similar results were observed with the downregulation of LdhA and pyruvate dehydrogenase kinase (Pdhk) isoenzymes 1, 2, and 3 in antibody producing‐CHO cells . However, the knockout of LdhA using zinc finger nucleases (ZFNs) in cells where Pdhk 1, 2, and 3 was down regulated was revealed to be lethal . The overexpression of Aralar1, part of malate–aspartate shuttle (MAS), in a lactate‐producing cell line led to a metabolic shift from lactate production to consumption .…”

Section: Cell Line Engineering For Improved Nutrient Metabolismsupporting

confidence: 54%

Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

Pereira

Kildegaard

Andersen

2018

Biotechnology Journal

152

146

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For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post-translational modifications. However, the metabolism of these cells is far from perfect and optimized, and requires substantial know how and process optimization and monitoring to perform efficiently. One of the main reasons for this is the production and accumulation of toxic and growth-inhibiting metabolites during culture. Lactate and ammonium are the most known, but many more have been identified. In this review, an overview of metabolites that deplete and accumulate throughout the course of cultivations with toxic and growth inhibitory effects to the cells is presented. Further, an overview of the CHO metabolism with emphasis to metabolic pathways of amino acids, glutathione (GSH), and related compounds which have growth-inhibiting and/or toxic effect on the cells is provided. Additionally, relevant publications which describe the applications of metabolomics as a powerful tool for revealing which reactions occur in the cell under certain conditions are surveyed and growth-inhibiting and toxic metabolites are identified. Also, a number of resources that describe the cellular mechanisms of CHO and are available on-line are presented. Finally, the application of this knowledge for bioprocess and medium development and cell line engineering is discussed.

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Section: Cell Line Engineering For Improved Nutrient Metabolismsupporting

confidence: 54%

Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

Pereira

Kildegaard

Andersen

2018

Biotechnology Journal

152

146

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“…Figure (A) shows point mutations highlighted in bold, the target sequence underlined and the restriction enzyme recognition site appears in italics. Results indicate that this particular clone could have three LDHa alleles because the ratios between Allele 1, Allele 2 and the Mutated Allele copies were approximately 1:1:1; the existence of an extra copy of LDHa has been reported previously by Yip and collaborators.…”

Section: Resultssupporting

confidence: 73%

An LDHa single allele CHO cell mutant exhibits altered metabolic state and enhanced culture performance

Wilkens

Vishwanathan

Baltes

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND Reducing lactate production in animal cell cultures has been reported to improve cell culture performance and productivity of recombinant protein. A novel genome editing tool, CRISPR/Cas, has been used widely to induce double‐strand breaks in the genome and introduce targeted mutations efficiently. RESULTS In the present work, we used a publicly available human‐codon optimized CRISPR/Cas system to introduce mutations in one of the LDHa gene copies to obtain an LDHa single‐allele knockout Chinese hamster ovary (CHO) cell clone to analyze its effect over cell metabolism. Fed‐batch cultures were conducted in order to evaluate the culture performance of mutant cells. Results show that cell growth was reduced and metabolism was modified by the LDHa single‐allele knockout, whereas specific protein rate and volumetric production were greatly enhanced. Additionally, the first in‐depth analysis of the metabolic effects of LDHa single allele knockout is presented. CONCLUSION By using CRISPR/Cas we were able to knock down LDHa, showing improved culture productivity. Metabolic flux analysis revealed higher fluxes in the TCA cycle in the mutant cell than in the parental cell, indicating that LDHa mutant cells have an enhanced energy metabolism, which can be attributed mainly to a higher catabolism of amino acids. © 2018 Society of Chemical Industry

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“…Therefore, after the subsequent induction, pyruvate reduces lactate production considerably for all cultures, reaching null presence of lactate in the medium. Lactate accumulation was observed after 24 h of induction, which is according to Yip et al, 29 who indicated that this situation can negatively impact cell growth during the fed-batch process. In this regard, the cultures at 2%M and 22 °C – 1.5%M and 18 °C had lower lactate concentration, but the latter had greater cell growth, while the rest of the cultures maintained a similar growth.…”

Section: Discussionmentioning

confidence: 66%

Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris

Zepeda

Figueroa

Pessoa

et al. 2018

Brazilian Journal of Microbiology

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The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 °C + 1%M provided the most stable viability. By contrast, the temperature of 18 °C + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.

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Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells

Cited by 16 publications

References 22 publications

Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

An LDHa single allele CHO cell mutant exhibits altered metabolic state and enhanced culture performance

Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris

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