TY - JOUR
T1 - Multiplex genome editing eliminates lactate production without impacting growth rate in mammalian cells
AU - Hefzi, Hooman
AU - Martínez-Monge, Iván
AU - Marin de Mas, Igor
AU - Cowie, Nicholas Luke
AU - Toledo, Alejandro Gomez
AU - Noh, Soo Min
AU - Karottki, Karen Julie la Cour
AU - Decker, Marianne
AU - Arnsdorf, Johnny
AU - Camacho-Zaragoza, Jose Manuel
AU - Kol, Stefan
AU - Schoffelen, Sanne
AU - Pristovšek, Nuša
AU - Hansen, Anders Holmgaard
AU - Miguez, Antonio A
AU - Bjørn, Sara Petersen
AU - Brøndum, Karen Kathrine
AU - Javidi, Elham Maria
AU - Jensen, Kristian Lund
AU - Stangl, Laura
AU - Kreidl, Emanuel
AU - Kallehauge, Thomas Beuchert
AU - Ley, Daniel
AU - Ménard, Patrice
AU - Petersen, Helle Munck
AU - Sukhova, Zulfiya
AU - Bauer, Anton
AU - Casanova, Emilio
AU - Barron, Niall
AU - Malmström, Johan
AU - Nielsen, Lars K
AU - Lee, Gyun Min
AU - Kildegaard, Helene Faustrup
AU - Voldborg, Bjørn G
AU - Lewis, Nathan E
N1 - © 2025. The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2025/1/14
Y1 - 2025/1/14
N2 - The Warburg effect, which describes the fermentation of glucose to lactate even in the presence of oxygen, is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production in cells for bioprocessing have failed as lactate dehydrogenase is essential for cell growth. Here, we effectively eliminate lactate production in Chinese hamster ovary and in the human embryonic kidney cell line HEK293 by simultaneous knockout of lactate dehydrogenases and pyruvate dehydrogenase kinases, thereby removing a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. These cells, which we refer to as Warburg-null cells, maintain wild-type growth rates while producing negligible lactate, show a compensatory increase in oxygen consumption, near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. Warburg-null cells remain amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titres and maintaining product glycosylation. The ability to eliminate lactate production may be useful for biotherapeutic production and provides a tool for investigating a common metabolic phenomenon.
AB - The Warburg effect, which describes the fermentation of glucose to lactate even in the presence of oxygen, is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production in cells for bioprocessing have failed as lactate dehydrogenase is essential for cell growth. Here, we effectively eliminate lactate production in Chinese hamster ovary and in the human embryonic kidney cell line HEK293 by simultaneous knockout of lactate dehydrogenases and pyruvate dehydrogenase kinases, thereby removing a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. These cells, which we refer to as Warburg-null cells, maintain wild-type growth rates while producing negligible lactate, show a compensatory increase in oxygen consumption, near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. Warburg-null cells remain amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titres and maintaining product glycosylation. The ability to eliminate lactate production may be useful for biotherapeutic production and provides a tool for investigating a common metabolic phenomenon.
U2 - 10.1038/s42255-024-01193-7
DO - 10.1038/s42255-024-01193-7
M3 - Article
C2 - 39809975
SN - 2522-5812
JO - Nature Metabolism
JF - Nature Metabolism
ER -