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Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.

Abstract：

: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.

journal_name

Biotechnol Bioeng

journal_title

Biotechnology and bioengineering

authors

Bulté DB,Palomares LA,Parra CG,Martínez JA,Contreras MA,Noriega LG,Ramírez OT

doi

10.1002/bit.27439

subject

Has Abstract

pub_date

2020-09-01 00:00:00

pages

2633-2647

issue

eissn

0006-3592

issn

1097-0290

journal_volume

117

pub_type

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Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.