Background Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. Results Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. Conclusion We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

Background Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. Results The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. Conclusions We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.

Itaconic acid is an emerging platform chemical with extensive applications. It is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen and needs additional morphology controls, and therefore the production remains problematic. Here, we reprogrammed the GRAS yeast Yarrowia lipolytica metabolism for competitive itaconic acid production. After redirecting the flux of lipid accumulation as carbon sink, we evaluated itaconic acid production both inside and outside the mitochondria, and fine modulated its synthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen replete conditions through down regulation of IDH by weak promoter changing, RNAi, or CRISPRi. Ultimately, we optimized fermentation parameters for fed-batch cultivations, and produced itaconic acid with titres of 130.1 g/L in 1L bioreactors and 94.8 g/L in a 50L bioreactor on semi-pilot scale. Our finds provide effective approaches for harnessing GRAS microorganism for competitive industrial itaconic acid production.

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Yarrowia lipolytica naturally saves carbon excess as storage lipids. Engineering efforts allow redirecting the high precursor flux required for lipid synthesis towards added-value chemicals such as polyketides, flavonoids, and terpenoids. To redirect precursor flux from storage lipids to other products, four genes involved in triacylglycerol and sterol ester synthesis (DGA1, DGA2, LRO1, ARE1) can be deleted. To elucidate the effect of the deletions on cell physiology and regulation, we performed chemostat cultivations under carbon and nitrogen limitation, followed by transcriptome analysis. We found that storage lipid-free cells show an enrichment of the unfolded protein response, and several biological processes related to protein refolding and degradation are enriched. Additionally, storage lipid-free cells show an altered lipid class distribution with an abundance of potentially cytotoxic free fatty acids under nitrogen limitation. Our findings not only highlight the importance of lipid metabolism on cell physiology and proteostasis, but can also aid the development of improved chassy strains of Y. lipolytica for commodity chemical production.