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Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

Hooman HefziIván Martínez-MongeIgor Marin de MasNicholas Luke CowieAlejandro Gomez ToledoSoo Min NohKaren Julie la Cour KarottkiMarianne DeckerJohnny ArnsdorfJose Manuel Camacho-ZaragozaStefan KolSanne SchoffelenNuša PristovšekAnders Holmgaard HansenAntonio A MiguezSara Petersen BjornKaren Kathrine BrøndumElham Maria JavidiKristian Lund JensenLaura StanglEmanuel KreidlThomas Beuchert KallehaugeDaniel LeyPatrice MénardHelle Munck PetersenZulfiya SukhovaAnton BauerEmilio CasanovaNiall BarronJohan MalmströmLars K NielsenGyun Min LeeHelene Faustrup KildegaardBjørn G VoldborgNathan E Lewis
Published in: bioRxiv : the preprint server for biology (2024)
The Warburg effect 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 via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.
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