[en] Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bulté, Dubhe B ; Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico
Palomares, Laura A ; Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico
Parra, Carolina Gómez; Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico
Martinez Alvarez, Juan Andrés ; Université de Liège - ULiège > Département GxABT > Microbial technologies ; UNAM - Universidad Nacional Autónoma de México [MX] > Instituto de Biotecnologia
Contreras, Martha A; Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico
Noriega, Lilia G; Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico
Ramírez, Octavio T ; Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico
Language :
English
Title :
Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.
Publication date :
September 2020
Journal title :
Biotechnology and Bioengineering
ISSN :
0006-3592
eISSN :
1097-0290
Publisher :
John Wiley and Sons Inc, Boschstrabe 12, 69469 Weinheim, Deutschland, United States
CONACYT - Consejo Nacional de Ciencia y Tecnología UNAM - Universidad Nacional Autónoma de México
Funding text :
The authors would like to acknowledge the following individuals for their contribution to this study: Dr. Mabel Rodríguez and Esmeralda Cuevas, MSc, for the plasmids donated for this study, Karina García for the EGFP‐expressing CHO cell line, Dr. Arturo Pimentel and National Laboratory of Microscopy for the support in the imaging analysis, Dr. Armando Trovar and the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán for the collaboration to perform the Seahorse experiments, Vanesa Hernández for technical support and Bernice Sandoval for her support during experiment execution. This project was financed by UNAM‐DGAPA‐PAPIIT IT‐200315, SEP‐CONACyT Ciencia Básica 255445 and CONACYT Infraestructura 280275. DBB was supported for her Ph.D. studies by a CONACYT scholarship. We are grateful to Laboratorios Liomont for providing the CHO‐S cell line used in this work.The authors would like to acknowledge the following individuals for their contribution to this study: Dr. Mabel Rodr?guez and Esmeralda Cuevas, MSc, for the plasmids donated for this study, Karina Garc?a for the EGFP-expressing CHO cell line, Dr. Arturo Pimentel and National Laboratory of Microscopy for the support in the imaging analysis, Dr. Armando Trovar and the Instituto Nacional de Ciencias M?dicas y Nutrici?n Salvador Zubir?n for the collaboration to perform the Seahorse experiments, Vanesa Hern?ndez for technical support and Bernice Sandoval for her support during experiment execution. This project was financed by UNAM-DGAPA-PAPIIT IT-200315, SEP-CONACyT Ciencia B?sica 255445 and CONACYT Infraestructura 280275. DBB was supported for her Ph.D. studies by a CONACYT scholarship. We are grateful to Laboratorios Liomont for providing the CHO-S cell line used in this work.
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