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.
Highlights
Yarrowia lipolytica is a non-conventional yeast with promising industrial potentials for lipids and citrate production
Y. lipolytica respiration is drastically decreased during citrate production phase and shifts from oxidative phosphorylation (OxPhos) to the alternative ΔΨ-independent pathway ensured by NADH2e/alternative oxidase (AOX) route
Isocitrate dehydrogenase and aconitase are predicted to operate in the backward direction and favor mitochondrial citrate accumulation
Summary
Yarrowia lipolytica is a non-conventional yeast with promising industrial potentials for lipids and citrate production It is widely used for studying mitochondrial respiration due to a respiratory chain like those of mammalian cells. Yarrowia lipolytica is a non-conventional yeast usually isolated from dairy products, oil and water e nvironments[1,2] This oleaginous yeast is emerging as an important cell factory for lipid production[3,4]. Y. lipolytica wild type strains isolated are Generally Recognized As Safe (GRAS) from the American Food and Drug Administration (FDA) and Qualified Presumption of Safety (QPS) from European Food Safety Authority (EFSA)[4,8,9] It is used as platform for the production of proteins (specially proteases and lipases)[2] and organic acids (citric acid, isocitric acid, acetic acid)[2,9,10]. Citrate and isocitrate are excreted to extracellular medium but citrate can be converted to acetyl-CoA, used as precursor for lipid s ynthesis[25,26]
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