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© 2018 Pomraning et al. The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae. We confirmed that the Y. lipolytica msn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica. Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1. Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain., Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica . To complement these strains, we constructed plasmids with Y. lipolytica -optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the " Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells., Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica . This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.

Background: Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield., Results: We found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation may be regulated by phosphorylation of key enzymes. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes., Conclusions: Integration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.

Yarrowia lipolytica is an oleaginous ascomycete yeast that accumulates large amounts of lipids and has potential as a biofuel producing organism. Despite a growing scientific literature focused on lipid production by Y. lipolytica, there remain significant knowledge gaps regarding the key biological processes involved. We applied a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica. We found that lipids accumulated rapidly and peaked at 48 hours during the five day experiment, concurrent with a shift in amino acid metabolism. We also report that exhaustion of extracellular sugars coincided with thickening of the cell wall, suggesting that genes involved in cell wall biogenesis may be a useful target for improving the efficiency of lipid producing yeast strains.