Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45°C) and the effect of oxidants (H₂O₂) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37°C) or treatment with a nonlethal dose of oxidants (0.5 mM H₂O₂) for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

Peroxisome biogenesis in the yeast Yarrowia lipolytica

Extensive perexisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis. A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast. The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glyco-sylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes. The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles. A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes. The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome. These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Serum-induced hypha formation in the dimorphic yeast Yarrowia lipolytica

The dimorphic yeast Yarrowia lipolytica forms true hyphae in a medium containing N-acetylglucosamine. We made a new finding that serum is a very effective inducer of hypha formation of Y. lipolytica: serum induced its hyphal growth very quickly compared to N-acetylglucosamine (4 h vs. 10 h). Osmotic and oxidative stresses (0.2 M NaCl and 20 mM H2O2) inhibited the hypha formation induced by N-acetylglucosamine, but did not suppress the hypha formation triggered by serum. Serum-specific morphological mutants, which formed hyphae in the N-acetylglucosamine medium but not in serum medium, could be isolated. These results suggest that the signal triggered by serum may be transduced through a different pathway, at least in part, from that used for the N-acetylglucosamine signal in Y. lipolytica.     

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

Double-stranded RNA from a marine alkane-degrading yeast Yarrowia lipolytica

A strain of Yarrowia lipolytica isolated from a tropical estuarine environment showed the presence of a double-stranded RNA. The double-stranded RNA had a molecular size of 5kb and a G+C content of 61 mole%. The strain was not a killer. No specific physiological function could be assigned to this plasmid.     

Double-stranded RNA from a marine alkane-degrading yeast Yarrowia lipolytica

A strain of Yarrowia lipolytica isolated from a tropical estuarine environment showed the presence of a double-stranded RNA. The double-stranded RNA had a molecular size of 5kb and a G+C content of 61 mole%. The strain was not a killer. No specific physiological function could be assigned to this plasmid.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21 %, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.     

Yarrowia lipolytica,a yeast genetic system to study mitochondrial complex I

The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I. A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized. The respiratory chain of Y. lipolytica contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX). Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y. lipolytica. Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side. Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene. Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y. lipolytica by affinity-chromatography. The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400-500 molecules of phosphatidylcholine per complex I. The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y. lipolytica complex I. Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.     

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.