The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K_m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.     

A Role for the Rap GTPase YlRsr1 in Cellular Morphogenesis and the Involvement of YlRsr1 and the Ras GTPase YlRas2 in Bud Site Selection in the Dimorphic Yeast Yarrowia lipolytica

Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for YlRSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of YlRSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1''s function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Ylras2Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2''s function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.     

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative α-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro α-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man₈GlcNAc₂, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man₈GlcNAc₂ to Man₁₂GlcNAc₂. Digestion with α-1,2- and α-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first α-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

Structural insight into the substrate specificity of acyl-CoA oxidase1 from Yarrowia lipolytica for short-chain dicarboxylyl-CoAs

Acyl-CoA oxidase (ACOX) plays an important role in fatty acid degradation. The enzyme catalyzes the first reaction in peroxisomal fatty acid β-oxidation by reducing acyl-CoA to 2-trans-enoyl-CoA. The yeast Yarrowia lipolytica is able to utilize fatty acids, fats, and oil as carbon sources to produce valuable bioproducts. We determined the crystal structure of ACOX1 from Y. lipolytica (YlACOX1) at a resolution of 2.5 Å. YlACOX1 forms a homodimer, and the monomeric structure is composed of four domains, the Nα, Nβ, Cα1, and Cα2. The FAD cofactor is bound at the dimerization interface between the Nβ- and Cα1-domains. The substrate-binding tunnel formed by the interface between the Nα-, Nβ-, and Cα1-domains is located proximal to FAD. Amino acid and structural comparisons of YlACOX1 with other ACOXs show that the substrate-binding pocket of YlACOX1 is much smaller than that of the medium- or long-chain ACOXs but is rather similar to that of the short-chain ACOXs. Moreover, the hydrophilicity of residues constituting the end region of the substrate-binding pocket in YlACOX1 is quite similar to those in the short-chain ACOXs but different from those of the medium- or long-chain ACOXs. These observations provide structural insights how YlACOX1 prefers short-chain dicarboxylyl-CoAs as a substrate.     

Yarrowia lipolytica dehydrogenase/reductase: An enzyme tolerant for lipophilic compounds and carbohydrate substrates

Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, purified and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose, suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance especially for space-demanding, lipophilic protecting groups, 5-O-trityl-d-ribitol and 5-O-trityl-α,β-d-ribose were investigated as substrates: YlSDR oxidized 5-O-trityl-d-ribitol and 5-O-trityl-α,β-d-ribose and reduced the latter at the expense of NADP(H).     

Regioselective hydrolysis of acetates in the presence of different yeast strains

The model compound, hexane-1,2-diol diacetate, was hydrolyzed in the presence of supernatant obtained after cultivation of 4 yeast strains: Pichia jadinii, Rhodotorula glutinis and Yarrowia lipolytica KKP 379 and Saccharomyces cerevisiae 102 to evaluate the type of catalysis. The regioselectivity of extracellular enzymes as a function of hydrolysis towards primary and secondary acetic acid ester groups was monitored. The enzymes secreted by P. jadinii, R. glutinis and Y. lipolytica KKP 379 exhibited high regioselectivity towards primary position, while those from S. cerevisiae showed practically no discrimination between the ester groups.     

Characterization of a lipid droplet protein from Yarrowia lipolytica that is required for its oleaginous phenotype

Oleaginous microorganisms are characterized by their ability to store high amounts of triacylglycerol (TAG) in intracellular lipid droplets (LDs). In this work, we characterized a protein of the oleaginous yeast Yarrowia lipolytica that is associated with LD and plays a role in the regulation of TAG storage. This protein is required for the oleaginous phenotype of Y. lipolytica because deletion of the coding gene results in a strongly reduced TAG content of the mutant. Therefore, we named it Oleaginicity Inducing LD protein, Oil1. Furthermore, a mutant overexpressing OIL1 accumulates more TAG than the wild type and is delayed in TAG lipolysis when this process is stimulated. We found that Oil1p plays a role in protecting the TAG content of the LD from degradation through lipases under conditions where the cell aims at building up its TAG reserves. Heterologous expression studies showed that Oil1p rescued the phenotype of a Saccharomyces cerevisiae mutant deleted for the perilipin-like protein Pln1p and that its expression in COS-7 cells resulted in increased TAG accumulation, similar to the phenotype of a perilipin 1 expressing control strain. Despite this phenotypical parallels to mammalian perilipins, Oil1p is not a member of this protein family and its activity does not depend on phosphorylation. Rather, our results suggest that ubiquitination might contribute to the function of Oil1p in Y. lipolytica and that a different mechanism evolved in this species to regulate TAG homeostasis.     

Construction of a whole-cell catalyst displaying a fungal lipase for effective treatment of oily wastewaters

To combine the advantage of the oleaginous yeast Yarrowia lipolytica with the high activity of some fungal lipases for oily wastewater treatment, an effective lipase-displaying arming yeast was constructed using the flocculation functional domain of Saccharomyces cerevisiae as the protein anchor. To estimate the effect of the whole-cell oily wastewater treatment, the lipase-displaying arming yeast was added into an open activated sludge bioreactor. Within 72 h of whole-cell treatment, 96.9% of oil and 97.6% of chemical oxygen demand (COD) were removed, while only 87.1% of oil and 91.8% of COD were removed in control A (Y. lipolytica Polg was added), 45.1% of oil and 67.5% of COD were removed in control B (no cell was added) in 72 h. The lipase-displaying arming yeast exhibited remarkable oil removal and COD degradation effect compared with the control samples, exemplifying its application potential.     

Dimorphism and hydrocarbon metabolism in Yarrowia lipolytica var. indica

Yarrowia lipolytica is able to metabolize high Mr hydrophobic natural compounds such as fatty acids and hydrocarbons. Characteristically, strains of Y. lipolytica can grow as populations with variable proportions of yeast and filamentous forms. In the present study, we describe the dimorphic characteristics of a variant designated as Y. lipolytica var. indica isolated from petroleum contaminated sea water and the effect of cell morphology on hydrocarbon metabolism. The variant behaved as a yeast monomorphic strain, under conditions at which terrestrial Y. lipolytica strain W29 and its derived strains, grow as almost uniform populations of mycelial cells. Using organic nitrogen sources and N-acetylglucosamine as carbon source, var. indica was able to form mycelial cells, the proportion of which increased when incubated under semi-anaerobic conditions. The cell surface characteristics of var. indica and W29 were found to be different with respect to contact angle and percent hydrophobicity. For instance, percent hydrophobicity of var. indica was 89.93 ± 1.95 while that of W29 was 70.78 ± 1.1. Furthermore, while all tested strains metabolize hydrocarbons, only var. indica was able to use it as a carbon source. Yeast cells of var. indica metabolized hexadecane with higher efficiency than the mycelial form, whereas the mycelial form of the terrestrial strain metabolized the hydrocarbon more efficiently, as occurred with the mycelial monomorphic mutant AC11, compared to the yeast monomorphic mutant AC1.     

Functional analysis of recombinant human and <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferases expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.