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.     

Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved

Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e.g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40 °C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60 °C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.     

Methyl Oleate Modulates <Emphasis Type="Italic">LIP2</Emphasis> Expression in the Lipolytic Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Methyl oleate was used as a primary carbon source and as an alternative inducer for the production of an extracellular lipase, Lip2, in Y. lipolytica strain LgX64.81 grown in a 20-l bioreactor. The lipase-encoding gene, LIP2, was investigated during culture on methyl oleate using a pLIP2–LacZ reporter fusion and we provide evidence for the involvement of methyl oleate in its regulation. Revisions requested 7 July 2005; Revisions received 30 August 2005     

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

We synthesized a Yarrowia lipolytica strain overproducing lipase for industrial applications by using long terminal repeat (ζ) of the Y. lipolytica retrotransposon Ylt1 and an allele of URA3 with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a NotI-NotI cassette which contains a defective URA3 allele, a polylinker sequence, and the ζ region for targeting to multiple sites in the genome of the recipient. We inserted the LIP2 gene (encoding extracellular lipase) under the control of the strong POX2 promoter into JMP3 to generate JMP6. The pHSS6 region was removed by NotI digestion prior to transformation. Two Y. lipolytica strains transformed with the JMP6 LIP2 cassette had a mean of 10 integrated copies devoid of the Escherichia coli region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the LIP2 promoter. This work provides a new expression system in Y. lipolytica that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.     

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2‐bromo phenyl acetic acid esters

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S‐enantioselectivity (E‐value = 5) during the hydrolytic kinetic resolution of 2‐bromo‐phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R‐selective enzyme (E‐value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low‐hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site‐directed mutagenesis. This work led to the isolation of one double substituted variant (D97A‐V232F), which displays a total preference for the R‐enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5‐fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R‐ and S‐enantiomers in the wild‐type enzyme and the D97A‐V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R‐enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity. Biotechnol. Bioeng. 2010;106: 852–859. © 2010 Wiley Periodicals, Inc.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.     

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.     

Expression,purification, crystallization,and diffraction analysis of a selenomethionyl lipase Lip8 from Yarrowia lipolytica

Yarrowia lipolytica is a nonconventional model micro-organism with multiple biotechnological applications. It is also considered to be an excellent producer for lipase. Genome survey shows that Y. lipolytica possesses various paralogs of genes coding for extracellular, cell-bound, and intracellular lipolytic enzymes. However, little structural information on these isoenzymes is available. With the aim to facilitate crystal structure solution of Lip8, one of the most valuable lipases from Y. lipolytica, a less conventional protein expression technique—selenomethionyl protein expression was used to produce recombinant selenomethionine (SeMet)-Lip8 in Escherichia coli. Finally, three Met residues of Lip8 were all substituted with SeMet. A total of 72?mg of SeMet-Lip8 was obtained from a liter of the SeMet medium. Using sodium acetate as a precipitant and ammonium sulfate as an additive, crystals of the SeMet-Lip8 with 1.9?Å were successfully cultured through hanging-drop vapor diffusion method. The estimated crystal dimensions were 0.11?×?0.11?×?0.14?mm². The crystal belonged to the space group I4 with unit cell parameters a?=?b?=?128.87?Å, c?=?171.77?Å, α?=?β?=?γ?=?90°. It is the second member of lipase crystal family from Y. lipolytica. This work will provide a platform for further studying lipases from a structural insight.     

Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut⁺ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression.     

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).     

Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling

Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man₈GlcNAc₂ to Man₅GlcNAc₂ efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.     

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.     

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.     

Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

A molecular approach to optimize hIFN α2b expression and secretion in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre–proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre–proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.     

Protein Production in Yarrowia lipolytica Via Fusion to the Secreted Lipase Lip2p

We established a strategy for protein production and purification via expression in Yarrowia lipolytica as Lip2p fusion protein. To evaluate the expression system a cysteine-rich miniprotein, an antibody fragment and an enzyme showing galactose oxidase activity were chosen. These proteins have varying disulfide bond content, size, and structural complexity. Endogenous lipase Lip2p was used as a fusion partner to direct the fused proteins to the extracellular medium. A linker sequence was introduced at the junction of Lip2p and the respective fused protein that contains a hexahistidine tag followed by a TEV protease cleavage site. This allows for a specific and simple purification via IMAC for capturing the secreted proteins from the supernatant followed by a second IMAC for removing all contaminants after proteolytic release of the protein of interest. Up to 174 mg/L fusion protein was obtained using shake flask cultivation. Functionality of each of the purified proteins was confirmed by individual assays. Expression of proteins of interest via Lip2p fusion not only provides a convenient expression and purification scheme but also enables for an online monitoring of accumulation of secreted fusion proteins in the medium by exploiting the intrinsic lipase activity of the fusion.     

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).