BackgroundYarrowia lipolytica is a non-conventional yeast that is extensively investigated for its ability to excrete citrate or to accumulate large amounts of storage lipids, which is of great significance for single cell oil production. Both traits are thus of interest for basic research as well as for biotechnological applications but they typically occur simultaneously thus lowering the respective yields. Therefore, engineering of strains with high lipid content relies on novel concepts such as computational simulation to better understand the two competing processes and to eliminate citrate excretion.ResultsUsing a genome-scale model (GSM) of baker's yeast as a scaffold, we reconstructed the metabolic network of Y. lipolytica and optimized it for use in flux balance analysis (FBA), with the aim to simulate growth and lipid production phases of this yeast. We validated our model and found the predictions of the growth behavior of Y. lipolytica in excellent agreement with experimental data. Based on these data, we successfully designed a fed-batch strategy to avoid citrate excretion during the lipid production phase. Further analysis of the network suggested that the oxygen demand of Y. lipolytica is reduced upon induction of lipid synthesis. According to this finding we hypothesized that a reduced aeration rate might induce lipid accumulation. This prediction was indeed confirmed experimentally. In a fermentation combining these two strategies lipid content of the biomass was increased by 80 %, and lipid yield was improved more than four-fold, compared to standard conditions.ConclusionsGenome scale network reconstructions provide a powerful tool to predict the effects of genetic modifications and the metabolic response to environmental conditions. The high accuracy and the predictive value of a newly reconstructed GSM of Y. lipolytica to optimize growth conditions for lipid accumulation are demonstrated. Based on these findings, further strategies for engineering Y. lipolytica towards higher efficiency in single cell oil production are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0217-4) contains supplementary material, which is available to authorized users.

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.

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or β) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Y. 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 S. 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.