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PubMed2006

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology.

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.

Antiporters↗
PubMed2004

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates.

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > MP > IMP. The apparent K(m) values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 degrees C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.

Adenosine Monophosphate↗
PubMed2005

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications.

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.

Alkanes↗
PubMed2005

Carbon source dependent phosphorylation of the Gpr1 protein in the yeast Yarrowia lipolytica.

The Gpr1 protein of the ascomycetous yeast Yarrowia lipolytica belongs to the poorly characterised Gpr1/Fun34/YaaH protein family whose members have been only found in prokaryotes and lower eukaryotes so far. Gpr1p seems to be involved in acetic acid adaptation at low pH values. Here we show that Gpr1p is subjected to phosphorylation in dependence on the carbon source. Exhaustion of the carbon source resulted in a complete dephosphorylation of Gpr1p, whereas addition of a new carbon source caused the phosphorylation of Gpr1p. Almost all Gpr1p molecules became phosphorylated after addition of acetate, while other carbon sources only triggered the phosphorylation of about half of the Gpr1p molecules. Phosphorylation was found to occur at serine-37. In spite of the clear effect of acetate/acetic acid on the level of phosphorylation of Gpr1p, no correlation of phosphorylation/dephosphorylation and acetic acid hypersensitivity, caused by mutations within Gpr1p, was detected.

Acetic Acid↗
PubMed2005

Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica.

In the lipolytic yeast Yarrowia lipolytica, the LIP2 gene was previously reported to encode an extracellular lipase. The growth of a Deltalip2 strain on triglycerides as sole carbon source suggest an alternative pathway for triglycerides utilisation in this yeast. Here, we describe the isolation and the characterisation of the LIP7 and LIP8 genes which were found to encode a 366 and a 371-amino acid precursor protein, respectively. These proteins which belong to the triacylglycerol hydrolase family (EC 3.1.1.3) presented a high homology with the extracellular lipase CdLIP2 and CdLIP3 from Candida deformans. The physiological function of the lipase isoenzymes was investigated by creating single and multi-disrupted strains. Lip7p and Lip8p were found to correspond to active secreted lipases. The lack of lipase production in a Deltalip2 Deltalip7 Deltalip8 strain suggest that no additional extracellular lipase remains to be discovered in Y. lipolytica. The substrate specificity towards synthetic ester molecules indicates that Lip7p presented a maximum activity centred on caproate (C6) while that of Lip8p is in caprate (C10).

Amino Acid Sequence↗
PubMed2006

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization.

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.

ATP-Binding Cassette Transporters↗
PubMed2004

Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review.

The production of heterologous proteins is a research field of high interest, with both academic and commercial applications. Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive. This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins. This review presents the main characteristics of Y. lipolytica, and the genetic and molecular tools available in this yeast. A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers. A table recapitulates the 42 heterologous proteins produced until now in Y. lipolytica. A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y. lipolytica physiology, and to offer a comparison with other production systems. This amount of data demonstrates the global reliability and versatility of Y. lipolytica as a host for heterologous production.

Gene Expression Regulation↗
PubMed2004

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica.

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.

Bioreactors↗
PubMed2006

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme.

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.

Cell Membrane↗
PubMed2003

New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica.

Yarrowia lipolytica is one of the most extensively studied nonconventional yeasts. Unfortunately, few methods for gene disruption have been reported for this yeast, and all of them are time-consuming and laborious. The functional analysis of unknown genes requires powerful disruption methods. Here, we describe such a new method for rapid gene disruption in Y. lipolytica. This knockout system combines SEP method and the Cre-lox recombination system, facilitating efficient marker rescue. Versatility was increased by using both auxotrophic markers like ylURA3 and ylLEU2, as well as the antibiotic resistance marker hph. The hph marker, which confers resistance to hygromycin-B, allows gene disruption in a strain lacking any conventional auxothrophic marker. The disruption cassette was shown to integrate at the correct locus at an average frequency of 45%. Upon expression of Cre recombinase, the marker was excised at a frequency of 98%, by recombination between the two lox sites. This new method for gene disruption is an ideal tool for the functional analysis of gene families, or for creating large-scale mutant collections in general.

DNA, Fungal↗
PubMed2006

Expression and purification of two lipases from Yarrowia lipolytica AS 2.1216.

We isolated two lipase genes LIPY7, LIPY8 from Yarrowia lipolytica CGMCC (China general microbiological culture collection center) AS 2.1216. The LIPY7 and LIPY8 genes encode a 366 and a 371-amino acid protein, respectively. The lipase genes with 6 x His tag sequence were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host Pichia pastoris KM71, respectively. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized.

Bacterial Proteins↗
PubMed2002

The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p.

One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.

Acyl Coenzyme A↗
PubMed2002

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.

Electron Spin Resonance Spectroscopy↗
PubMed2002

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids.

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 micromol NADH min(-1) mg(-1) were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.

Electron Transport Complex I↗
PubMed2002

YlALK1 encoding the cytochrome P450ALK1 in Yarrowia lipolytica is transcriptionally induced by n-alkane through two distinct cis-elements on its promoter.

The YlALK1 gene, which encodes cytochrome P450ALK1, plays a primary role in the assimilation of n-decane by yeast Yarrowia lipolytica and is inducible by n-decane at the transcriptional level. Deletion analysis of the YlALK1 promoter revealed that a 95-bp region on the YlALK1 promoter (from the position -400 to -304 upstream of the ATG codon) is essential for the induction by n-decane and we named this region ARR1 (alkane-responsive region). ARR1 was found to be made up of two different elements, ARE1 (alkane-responsive element 1; from -394 to -371) and ARE2 (from -325 to -305). By electrophoretic mobility shift assay, we found that the respective elements gave specific shift bands with the extracts from Y. lipolytica cells grown on n-alkane, but not much evidently from the cells grown on glycerol or glucose. This suggests that proteins that specifically bind to these elements are present and their binding or synthesis is dependent on n-alkane.

Alkanes↗
PubMed2002

Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single-stage continuous culture.

Yarrowia lipolytica LGAM S(7)1 presented remarkable growth on industrial glycerol used as sole carbon substrate. Nitrogen-limited flask cultures were accompanied by restricted synthesis of reserve lipid, whilst amounts of citric acid were produced extracellularly. On the contrary, high amounts of reserve lipid (up to 3.5 g/l, 43% w/w of lipids in dry biomass) were produced in highly aerated continuous cultures. Lipid production was favoured at low specific dilution rates whilst fat-free material yield increased over the whole range of D (h(-1)). The maximum volumetric productivity obtained was 0.12 g lipid/1 h. Storage lipid composition did not present remarkable changes in the specific dilution rates tested. Oleate and linoleate were the dominant cellular fatty acids.

Biomass↗
PubMed2002

Protein expression and secretion in the yeast Yarrowia lipolytica.

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.

Bacterial Proteins↗
PubMed2003

Oxygen requirements for growth and citric acid production of Yarrowia lipolytica.

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.

Aconitate Hydratase↗