Fine-tuning mitochondrial activity in Yarrowia lipolytica for citrate overproduction

Abstract

Yarrowia lipolytica is a non-conventional yeast with promising industrial potentials for lipids and citrate production. It is also widely used for studying mitochondrial respiration due to a respiratory chain like those of mammalian cells. In this study we used a genome-scale model (GEM) of Y. lipolytica metabolism and performed a dynamic Flux Balance Analysis (dFBA) algorithm to analyze and identify metabolic levers associated with citrate optimization. Analysis of fluxes at stationary growth phase showed that carbon flux derived from glucose is rewired to citric acid production and lipid accumulation, whereas the oxidative phosphorylation (OxPhos) shifted to the alternative respiration mode through alternative oxidase (AOX) protein. Simulations of optimized citrate secretion flux resulted in a pronounced lipid oxidation along with reactive oxygen species (ROS) generation and AOX flux inhibition. Then, we experimentally challenged AOX inhibition by adding n-Propyl Gallate (nPG), a specific AOX inhibitor, on Y. lipolytica batch cultures at stationary phase. Our results showed a twofold overproduction of citrate (20.5 g/L) when nPG is added compared to 10.9 g/L under control condition (no nPG addition). These results suggest that ROS management, especially through AOX activity, has a pivotal role on citrate/lipid flux balance in Y. lipolytica. All taken together, we thus provide for the first time, a key for the understanding of a predominant metabolic mechanism favoring citrate overproduction in Y. lipolytica at the expense of lipids accumulation.

A Review on Citric Acid Production by Yarrowia lipolytica Yeast: Past and Present Challenges and Developments

“…However, when cultivated in glucose-enriched olive mill wastewater, the strain produced only 15.8 g/L CA with a yield (Y CA ) of 0.46 g/g [116]. Using this particular strain, it was shown that the control of reactive oxygen species, particularly via alternative oxidase activity, is essential for regulating the balance between citrate and lipid fluxes [118].…”

Combining Kinetic and Constraint-Based Modelling to Better Understand Metabolism Dynamics

“…Those different versions of dFBA have been widely used to study the dynamics of metabolism. For instance, dFBA has been used to study the optimisation of citrate production in Yarrowia lipolytica [42]. In this article, da Veiga Moreira et al apply the SOA approach on a genome-scale model (about 2000 reactions and metabolites) and find that by adding n-Propyl Gallate, an inhibitor of the alternative oxidase, the production of citrate is substantially increased.…”