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PubMed2003

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster.

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590.

Aldose-Ketose Isomerases↗
PubMed2003

Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 Delta strain.

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.

Amino Acid Sequence↗
PubMed2002

Different effectors of dimorphism in Yarrowia lipolytica.

Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches.

Carbon↗
PubMed2001

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions.

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying H(+)- and Na(+)-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5-10.0. In cells grown at pH 4.5, inorganic phosphate (P(i)) was accumulated by two kinetically discrete H(+)/P(i)-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P(i) concentrations. The high-affinity system, subjected to regulation by both extracellular P(i) availability and intracellular polyphosphate stores, is mobilized during P(i)-starvation. In cells grown at pH 9.5-10, P(i) uptake is mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P(i) concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P(i)-starvation and appear to be controlled by extracellular P(i). They represent the first examples of high-capacity, Na(+)-driven P(i) transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H(+)- and Na(+)-coupled P(i) transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H(+)-coupled P(i) transport systems are predominant. The contribution of the Na(+)/P(i) cotransport systems to the total cellular P(i) uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.

Acids↗
PubMed2001

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures.

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.

Air Movements↗
PubMed2002

Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation.

The effect of ethanol, zinc, and iron (Fe(2+) and Fe(3+)) concentration and of oxygen supply on cell growth and the production of citric acid (CA) and isocitric acid (ICA) from ethanol by mutant Yarrowia lipolytica N 1 was studied under continuous cultivation. The following peculiarities of Y. lipolytica metabolism were found: (1) intensive CA production occurred under yeast growth limitation by nitrogen; (2) inhibition of yeast growth by ethanol was accompanied by significant alterations in fatty acid composition of lipids; (3) the production of CA and ICA from ethanol required high concentrations of zinc and iron ions; (4) the intracellular iron concentration determined whether CA or ICA was predominantly formed; (5) the cell's requirement for oxygen depended on the intracellular iron concentration. The events taking place in the production of CA and ICA were evaluated through the activities of enzyme systems involved in the metabolism of ethanol and CA in this strain.

Citric Acid↗
PubMed2003

Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica.

The gamma- and delta-lactones of less than 12 carbons constitute a group of compounds of great interest to the flavour industry. It is possible to produce some of these lactones through biotechnology. For instance, gamma-decalactone can be obtained by biotransformation of methyl ricinoleate. Among the organisms used for this bioproduction, Yarrowia lipolytica is a yeast of choice. It is well adapted to growth on hydrophobic substrates, thanks to its efficient and numerous lipases, cytochrome P450, acyl-CoA oxidases and its ability to produce biosurfactants. Furthermore, genetic tools have been developed for its study. This review deals with the production of lactones by Y. lipolytica with special emphasis on the biotransformation of methyl ricinoleate to gamma-decalactone. When appropriate, information from the lipid metabolism of other yeast species is presented.

Biotechnology↗
PubMed2003

Overproduction of lipase by Yarrowia lipolytica mutants.

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2- LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.

Biotechnology↗
PubMed2004

Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications.

Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.

Base Sequence↗
PubMed2006

Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica.

There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l(-1) after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l(-1) of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)(-1)] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)(-1)]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.

Benzoate 4-Monooxygenase↗
PubMed2006

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180.

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35 degrees C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of < or =10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.

Amino Acid Sequence↗
PubMed2003

Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues.

Yarrowia lipolytica was cultivated on mixtures of saturated free fatty acids (an industrial derivative of animal fat called stearin), technical glycerol (the main by-product of bio-diesel production facilities), and glucose. The utilization of technical glycerol and stearin as co-substrates resulted in higher lipid synthesis and increased citric acid production than the combination of glucose and stearin. The lipids produced contained significant amounts of stearic acid (50-70%, wt/wt) and lower ones of palmitic (15-20%, wt/wt), oleic (7-20%, wt/wt), and linoleic (2-7%, wt/wt) acid. Single-cell oil having a composition similar to cocoa-butter up to 3.4 g/L was produced, whereas in some cases relatively increased citric acid quantities (up to 14 g/L) were excreted into the growth medium. The microorganism presented a high specificity for lauric, myristic, and palmitic acid, while a discrimination for the stearic acid was observed. As a conclusion, microbial metabolism could be directed by using mixtures of inexpensive saturated fats, glycerol, and glucose as co-substrates, in order to accumulate lipids with predetermined composition, e.g., cocoa-butter equivalents.

Citric Acid↗
PubMed2003

Modeling lipid accumulation and degradation in Yarrowia lipolytica cultivated on industrial fats.

A modeling approach was used to quantify the kinetic behavior of a Yarrowia lipolytica strain capable of producing significant lipid amounts when cultivated on industrial fats. Biomass and cellular lipid evolution were successfully simulated, while the optimized parameter values were similar to those experimentally measured. The maximum specific formation rate of fat-free biomass seemed unaffected by the substrate fatty acid composition. On the contrary, the maximum concentration of lipid accumulated inside the yeast cell, as well as the maximum specific accumulation rate of cellular lipids, was favored in high stearic acid content media. The microorganism presented the tendency to degrade its accumulated lipids, although remarkable substrate fat amounts remained unconsummated in the culture medium. This degradation slowly occurred in the yeast cell as the specific rate of the intracellular carbon pool (storage lipid consumption) was significantly lower compared with that of the extracellular carbon pool (substrate fat). However, the fat-free biomass yield on storage lipids (g of fat-free biomass formed per g of storage lipids consumed) was higher than the one on the substrate (g of fat-free biomass formed per g of medium fat consumed).

Biomass↗
PubMed2005

Involvement of hexokinase Hxk1 in glucose catabolite repression of LIP2 encoding extracellular lipase in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.

Bacterial Proteins↗
PubMed2006

The ambient pH response Rim pathway in Yarrowia lipolytica: identification of YlRIM9 and characterization of its role in dimorphism.

Yarrowia lipolytica is a dimorphic fungus that secretes either an acidic or an alkaline protease depending on the environmental pH. Previous results have indicated that secretion of the alkaline protease is under control of the pH signaling Pal/Rim pathway originally described in Aspergillus nidulans. Several Y. lipolytica mutants defective in some Rim components of this pathway have been previously isolated and the RIM genes characterized. In the present study, Y. lipolytica RIM9 (palI) gene (YlRIM9) was sequenced from a plasmid (AL414126) of the Genolevures project (the DNA sequence data for YlRIM9 gene has been deposited at EMBL with accession number AJ566902). The derived translation product contains 724 amino acids with a predicted signal peptide and four transmembrane domains in its N-terminal region. We demonstrated that mutation in YlRIM9, as well as in other genes encoding members of the Pal/Rim pathway, did not affect the pH-dependent dimorphic transition of Y. lipolytica. A different pathway must exist in this fungus that controls the effect of pH on dimorphism.

Fungal Proteins↗
PubMed2001

Factors affecting the morphogenetic switch in Yarrowia lipolytica.

Yarrowia lipolytica is a dimorphic yeast usually isolated from dairy products. Here we described methods for inducing in a homogeneous way a true yeast-hypha transition in liquid medium. As a first step, the cells must be synchronized in the G1 phase of the cell cycle by nitrogen starvation. Using either N-acetylglucosamine (GlcNAc) or serum as the only carbon sources, more than 90% of the cells form hypha after 4-6 h of incubation. Bovine albumin is also able to induce the yeast-hypha transition, although to a lesser extent. The addition of glucose to cultures growing with GlcNAc arrest the morphogenetic switch but not when added to cultures growing in the presence of serum. Serum also induces invasive growth in solid medium. Neither pH, nitrogen starvation, nor temperature play a relevant role in the morphogenetic switch. Our results suggest that, as occurs in Candida albicans, at least two morphogenetic signal pathways exist in Y. lipolytica.

Acetylglucosamine↗
PubMed2006

Two Hrd1p homologues in the yeast Yarrowia lipolytica which act in different pathways.

The endoplasmic reticulum associated degradation (ERAD) is a process widespread in eukaryotes that enable cells to get rid of unfolded or unassembled polypeptides which jam the endoplasmic reticulum compartment. In order to improve understanding of the initial steps of the secretory pathway and their relationship, we focused on components of the ERAD ubiquitylation machinery in the yeast Yarrowia lipolytica. Two Hrd1p homologues, Hrd1p and Hrh1p, were identified in Y. lipolytica. A study of the fate of the heterologous CPY* reporter protein showed that YlHrd1p is involved in the elimination of this misfolded polypeptide, while YlHrh1p is not. Moreover, the different phenotypic pattern displayed by Deltahrd1 and Deltahrh1 cells suggests that the two putative E3 enzymes function in separate ways. Our results bring some evidence of a coupling between the ERAD pathway and the co-translational translocation process and show that studies in Y. lipolytica can give new insights into events that take place in the ER.

Amino Acid Sequence↗
PubMed2006

Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy.

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.

Electron Spin Resonance Spectroscopy↗