Sugar versus fat: elimination of glycogen storage improves lipid accumulation in Yarrowia lipolytica

Abstract

Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Yarrowia lipolytica and found that this yeast accumulates up to 16% glycogen in its biomass. Assuming that elimination of glycogen synthesis would result in an improvement of lipid accumulation, we characterized and deleted the single gene coding for glycogen synthase, YlGSY1. The mutant was grown under lipogenic conditions with glucose and glycerol as substrates and we obtained up to 60% improvement in TAG accumulation compared to the wild-type strain. Additionally, YlGSY1 was deleted in a background that was already engineered for high lipid accumulation. In this obese background, TAG accumulation was also further increased. The highest lipid content of 52% was found after 3 days of cultivation in nitrogen-limited glycerol medium. Furthermore, we constructed mutants of Y. lipolytica and Saccharomyces cerevisiae that are deleted for both glycogen and TAG synthesis, demonstrating that the ability to store carbon is not essential. Overall, this work showed that glycogen synthesis is a competing pathway for TAG accumulation in oleaginous yeasts and that deletion of the glycogen synthase has beneficial effects on neutral lipid storage.

Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica

“…A recent work showed that disruption of glycogen synthase ( GSY1 ) in Y. lipolytica gave an increase in lipid accumulation by diverting carbon from carbohydrate storage to lipid storage ( Bhutada et al, 2017 ). We hypothesized that eliminating glycogen biosynthesis may increase overall acetyl-CoA production, and that some of this increased pool of acetyl-CoA might then enter the mevalonate pathway and lead to higher lycopene yields.…”

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

“…Detection of glycogen synthase activity using Lugol's solution. When yellow wells were present, it indicated a defect in glycogen synthase, which is a characteristic of the Dgsy1 mutant strain (Bhutada et al, 2017). In this case, the JMY1212 strain was used to test the integration at gsy locus.…”

Identification of a Δ11 desaturase from the arbuscular mycorrhizal fungusRhizophagus irregularis

“…The open reading frames of DES1 and DES2 were codon optimised for expression in S. cerevisiae by Genscript, synthesised and supplied in the pUC-57 vector. DES1 and DES2 were then excised using BamHI and SalI restriction sites and ligated into pHEY1 [22], for expression under the constitutive TEF1 promoter. pHEY-DES1, pHEY-DES2 and pHEY-EVC (empty vector) were transformed into S. cerevisiae [23] wild type strain DTY-11a (MATa, leu2-3, leu2-12, trp1-1, can1-100, ura3-1, ade2-1) and ole1Δ knockout strain AMY-3α (MATα, ole1Δ::LEU2, trp1-1, can1-100, ura3-1, ade2-1) [24] and colonies were selected on synthetic Dexterose (SD) minimal medium agar plates lacking uracil.…”

“…To test the enzymatic function of DES1 and DES2 we transformed wild type S. cerevisiae and the desaturation-deficient ole1Δ knock-out strain [24,29] with the high copy number plasmids pHEY-DES1 and pHEY-DES2, designed to express the two genes under the control of the strong constitutive translational elongation factor EF-1α (TEF1) promoter [22]. The ole1Δ strain is completely deficient in fatty acid desaturation and can only grow on media that is supplemented with exogenous long-chain unsaturated fatty acids [24,29].…”