Transient vitamin B5 starving improves mammalian cell homeostasis and protein production

Lucille Pourcel,Flavien Buron,Fanny Garcia,Margaux-Sarah Delaloix,Valérie Le Fourn,Pierre-Alain Girod,Nicolas Mermod

doi:10.1016/j.ymben.2020.03.008

Abstract

Maintaining a metabolic steady state is essential for an organism's fitness and survival when confronted with environmental stress, and metabolic imbalance can be reversed by exposing the organism to fasting. Here, we attempted to apply this physiological principle to mammalian cell cultures to improve cellular fitness and consequently their ability to express recombinant proteins. We showed that transient vitamin B5 deprivation, an essential cofactor of central cellular metabolism, can quickly and irreversibly affect mammalian cell growth and division. A selection method was designed that relies on mammalian cell dependence on vitamin B5 for energy production, using the co-expression of the B5 transporter SLC5A6 and a gene of interest. We demonstrated that vitamin B5 selection persistently activates peroxisome proliferator-activated receptors (PPAR), a family of transcription factors involved in energy homeostasis, thereby altering lipid metabolism, improving cell fitness and therapeutic protein production. Thus, stable PPAR activation may constitute a cellular memory of past deprivation state, providing increased resistance to further potential fasting events. In other words, our results imply that cultured cells, once exposed to metabolic starvation, may display an improved metabolic fitness as compared to non-exposed cells, allowing increased resistance to cellular stress.

Highlights

Large amounts of active protein are usually required for functional and structural analysis, as well as for the production of biological therapeutics
We first aimed to capitalize on mammalian cell dependence on vitamin B5 for central metabolism balance, in order to select cells with improved fitness and energy homeostasis, as required to efficiently express complex therapeutic proteins
We provide evidence that cells transiently deprived of vitamin B5 may have acquired persistent alterations in their lipid metabolism, as mediated by the activation of PPARα transcription factor

Summary

Introduction

Large amounts of active protein are usually required for functional and structural analysis, as well as for the production of biological therapeutics. The liver maintains energy homeostasis during fasting by activation of ketogenesis, to provide enough energy-yielding nutrients for critical organs such as brain and muscles (van den Berghe, 1991). In such conditions, the hepatic acetyl-CoA, a key molecule in central metabolism, will be converted into ketones instead of entering the TCA cycle (Pietrocola et al, 2015). The hepatic acetyl-CoA, a key molecule in central metabolism, will be converted into ketones instead of entering the TCA cycle (Pietrocola et al, 2015) These ketones will enter blood circulation to be taken up into extra-hepatic cells, reconverted into acetyl-CoA and metabolized by the TCA cycle for ATP production (Puchalska and Crawford, 2017)

Methods

Results

Conclusion