Gene_locus Report for: yarli-lip2

ESTHER: Zhang_2019_Int.J.Biol.Macromol_137_1190
PubMedSearch: Zhang 2019 Int.J.Biol.Macromol 137 1190
PubMedID: 31299254
Gene_locus related to this paper: yarli-lip2

Abstract

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 degrees C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 degrees C, which was about 5.0 degrees C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.

ESTHER: Dujon_2004_Nature_430_35
PubMedSearch: Dujon 2004 Nature 430 35
PubMedID: 15229592
Gene_locus related to this paper: canga-apth1, canga-ppme1, canga-q6fik7, canga-q6fiv5, canga-q6fiw8, canga-q6fj11, canga-q6fjh6, canga-q6fjl0, canga-q6fjr8, canga-q6fkj6, canga-q6fkm9, canga-q6fku7, canga-q6fl14, canga-q6flb5, canga-q6fle9, canga-q6flk8, canga-q6fly1, canga-q6fly9, canga-q6fmz4, canga-q6fnx4, canga-q6fp28, canga-q6fpa8, canga-q6fpi6, canga-q6fpv7, canga-q6fpw6, canga-q6fqj3, canga-q6fr97, canga-q6frt7, canga-q6ftm9, canga-q6ftu0, canga-q6ftv9, canga-q6ftz9, canga-q6fuf8, canga-q6fv41, canga-q6fvu3, canga-q6fw36, canga-q6fw94, canga-q6fwk6, canga-q6fwm0, canga-q6fxc7, canga-q6fxd7, debha-apth1, debha-atg15, debha-b5rtk1, debha-b5rub4, debha-b5rue8, debha-b5rue9, debha-bna7, debha-ppme1, debha-q6bgx3, debha-q6bh69, debha-q6bhb8, debha-q6bhc1, debha-q6bhd0, debha-q6bhj7, debha-q6bi97, debha-q6biq7, debha-q6bj53, debha-q6bkd8, debha-q6bks1, debha-q6bky4, debha-q6bm63, debha-q6bmh3, debha-q6bn89, debha-q6bnj6, debha-q6bp08, debha-q6bpb4, debha-q6bpc0, debha-q6bpc6, debha-q6bq10, debha-q6bq11, debha-q6bqd9, debha-q6bqj6, debha-q6br33, debha-q6br93, debha-q6brg1, debha-q6brw7, debha-q6bs23, debha-q6bsc3, debha-q6bsl8, debha-q6bsx6, debha-q6bta5, debha-q6bty5, debha-q6btz0, debha-q6bu73, debha-q6buk9, debha-q6but7, debha-q6bvc4, debha-q6bvg4, debha-q6bvg8, debha-q6bvp4, debha-q6bw82, debha-q6bxr7, debha-q6bxu9, debha-q6bym5, debha-q6byn7, debha-q6bzj8, debha-q6bzk2, debha-q6bzm5, klula-apth1, klula-ppme1, klula-q6cin9, klula-q6ciu6, klula-q6cj47, klula-q6cjc8, klula-q6cjq9, klula-q6cjs1, klula-q6cjv9, klula-q6ckd7, klula-q6ckk4, klula-q6ckx4, klula-q6cl20, klula-q6clm1, klula-q6cly8, klula-q6clz7, klula-q6cm48, klula-q6cm49, klula-q6cmt5, klula-q6cn71, klula-q6cnm1, klula-q6cr74, klula-q6cr90, klula-q6crs0, klula-q6crv8, klula-q6crz9, klula-q6cst8, klula-q6csv8, klula-q6ctp8, klula-q6cu02, klula-q6cu78, klula-q6cu79, klula-q6cuv3, klula-q6cvd3, klula-q6cw70, klula-q6cw92, klula-q6cwu7, klula-q6cx84, klula-q6cxa3, klula-q6cy41, yarli-apth1, yarli-atg15, yarli-BST1B, yarli-lip2, yarli-LIP3, yarli-LIP4, yarli-LIP5, yarli-LIP7, yarli-LIP8, yarli-lipa1, yarli-ppme1, yarli-q6bzp1, yarli-q6bzv7, yarli-q6c1f5, yarli-q6c1f7, yarli-q6c1r3, yarli-q6c2z2, yarli-q6c3h1, yarli-q6c3i6, yarli-q6c3l1, yarli-q6c3u6, yarli-q6c4h8, yarli-q6c5j1, yarli-q6c5m4, yarli-q6c6m4, yarli-q6c6p7, yarli-q6c6v2, yarli-q6c7h3, yarli-q6c7i7, yarli-q6c7j5, yarli-q6c7y6, yarli-q6c8m4, yarli-q6c8q4, yarli-q6c8u4, yarli-q6c8y2, yarli-q6c9r0, yarli-q6c9r1, yarli-q6c9u0, yarli-q6c9v4, yarli-q6c209, yarli-q6c225, yarli-q6c493, yarli-q6c598, yarli-q6c687, yarli-q6c822, yarli-q6cau6, yarli-q6cax2, yarli-q6caz1, yarli-q6cb63, yarli-q6cba7, yarli-q6cbb1, yarli-q6cbe6, yarli-q6cby1, yarli-q6ccr0, yarli-q6cdg1, yarli-q6cdi6, yarli-q6cdv9, yarli-q6ce37, yarli-q6ceg0, yarli-q6cep3, yarli-q6cey5, yarli-q6cf60, yarli-q6cfp3, yarli-q6cfx2, yarli-q6cg13, yarli-q6cg27, yarli-q6cgj3, yarli-q6chb8, yarli-q6ci59, yarli-q6c748, canga-q6fpj0, klula-q6cp11, yarli-q6c4p0, debha-q6btp5, debha-kex1

Abstract

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

ESTHER: Pignede_2000_J.Bacteriol_182_2802
PubMedSearch: Pignede 2000 J.Bacteriol 182 2802
PubMedID: 10781549
Gene_locus related to this paper: aspnc-a5abh9, yarli-lip2

Abstract

We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2(A) (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica..

ESTHER: Zhang_2019_Int.J.Biol.Macromol_137_1190
PubMedSearch: Zhang 2019 Int.J.Biol.Macromol 137 1190
PubMedID: 31299254
Gene_locus related to this paper: yarli-lip2

Abstract

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 degrees C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 degrees C, which was about 5.0 degrees C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.

ESTHER: Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
PubMedSearch: Aloulou 2013 Eur.J.Lipid.Sci.Tech 115 429
Gene_locus related to this paper: yarli-lip2

Abstract

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) is assumed to be a good drug candidate for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. Understanding and improving its biochemical properties are essential for its oral administration. YLLIP2 is a highly glycosylated protein, with glycan chains accounting for about 13% of the molecular mass of the native protein. Two potential N-glycosylation sites (N113IS and N134NT) can be identified from YLLIP2 amino acid sequence. YLLIP2 mutants with single (N113Q or N134Q) or combined (N113Q/N134Q) substitutions of these glycosylation sites were expressed in the yeast Pichia pastoris, purified and characterized. Lipase specific activity and adsorption at the lipidwater interface were found to be decreased in the absence of N-glycosylation. It was thus shown that the glycosylated enzyme had a better ability to bind and penetrate a DLPC monolayer than the non-glycosylated N113Q/N134Q mutant. Comparison of wild-type glycosylated and non-glycosylated YLLIP2 shows that the N-glycosylation clearly contributes to the high stability of YLLIP2 in the presence of pepsin in vitro, and to a lower extent in the presence of chymotrypsin. The X-ray structure of the YLLIP2 N113Q/N134Q double mutant was obtained at 2.6 angstrom resolution and was found to be identical to that of wild-type YLLIP2, with the lid in a closed conformation. Glycosylation is therefore not essential for a proper folding of YLLIP2. Practical applications: The LIP2 lipase from the yeast Yarrowia lipolytica is one of the most active lipases identified so far. Among the various applications envisioned for this enzyme, it seems particularly well adapted for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. It is active and stable at low pH values, resistant to bile salts, and its glycosylation allows a high resistance to pepsin. All these properties are important for developing the oral administration of digestive enzymes used as drugs.

ESTHER: Darvishi_2011_N.Biotechnol_28_756
PubMedSearch: Darvishi 2011 N.Biotechnol 28 756
PubMedID: 21324386
Gene_locus related to this paper: yarli-lip2

Abstract

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37 degrees C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.

ESTHER: Bordes_2010_Biophys.J_99_2225
PubMedSearch: Bordes 2010 Biophys.J 99 2225
PubMedID: 20923657
Gene_locus related to this paper: yarli-lip2

Abstract

We report the 1.7 A 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.

ESTHER: Cambon_2010_Biotechnol.Bioeng_106_852
PubMedSearch: Cambon 2010 Biotechnol.Bioeng 106 852
PubMedID: 20506522
Gene_locus related to this paper: yarli-lip2

Abstract

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.

ESTHER: Bordes_2009_Chembiochem_10_1705
PubMedSearch: Bordes 2009 Chembiochem 10 1705
PubMedID: 19504508
Gene_locus related to this paper: yarli-lip2

Abstract

Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.

ESTHER: Dujon_2004_Nature_430_35
PubMedSearch: Dujon 2004 Nature 430 35
PubMedID: 15229592
Gene_locus related to this paper: canga-apth1, canga-ppme1, canga-q6fik7, canga-q6fiv5, canga-q6fiw8, canga-q6fj11, canga-q6fjh6, canga-q6fjl0, canga-q6fjr8, canga-q6fkj6, canga-q6fkm9, canga-q6fku7, canga-q6fl14, canga-q6flb5, canga-q6fle9, canga-q6flk8, canga-q6fly1, canga-q6fly9, canga-q6fmz4, canga-q6fnx4, canga-q6fp28, canga-q6fpa8, canga-q6fpi6, canga-q6fpv7, canga-q6fpw6, canga-q6fqj3, canga-q6fr97, canga-q6frt7, canga-q6ftm9, canga-q6ftu0, canga-q6ftv9, canga-q6ftz9, canga-q6fuf8, canga-q6fv41, canga-q6fvu3, canga-q6fw36, canga-q6fw94, canga-q6fwk6, canga-q6fwm0, canga-q6fxc7, canga-q6fxd7, debha-apth1, debha-atg15, debha-b5rtk1, debha-b5rub4, debha-b5rue8, debha-b5rue9, debha-bna7, debha-ppme1, debha-q6bgx3, debha-q6bh69, debha-q6bhb8, debha-q6bhc1, debha-q6bhd0, debha-q6bhj7, debha-q6bi97, debha-q6biq7, debha-q6bj53, debha-q6bkd8, debha-q6bks1, debha-q6bky4, debha-q6bm63, debha-q6bmh3, debha-q6bn89, debha-q6bnj6, debha-q6bp08, debha-q6bpb4, debha-q6bpc0, debha-q6bpc6, debha-q6bq10, debha-q6bq11, debha-q6bqd9, debha-q6bqj6, debha-q6br33, debha-q6br93, debha-q6brg1, debha-q6brw7, debha-q6bs23, debha-q6bsc3, debha-q6bsl8, debha-q6bsx6, debha-q6bta5, debha-q6bty5, debha-q6btz0, debha-q6bu73, debha-q6buk9, debha-q6but7, debha-q6bvc4, debha-q6bvg4, debha-q6bvg8, debha-q6bvp4, debha-q6bw82, debha-q6bxr7, debha-q6bxu9, debha-q6bym5, debha-q6byn7, debha-q6bzj8, debha-q6bzk2, debha-q6bzm5, klula-apth1, klula-ppme1, klula-q6cin9, klula-q6ciu6, klula-q6cj47, klula-q6cjc8, klula-q6cjq9, klula-q6cjs1, klula-q6cjv9, klula-q6ckd7, klula-q6ckk4, klula-q6ckx4, klula-q6cl20, klula-q6clm1, klula-q6cly8, klula-q6clz7, klula-q6cm48, klula-q6cm49, klula-q6cmt5, klula-q6cn71, klula-q6cnm1, klula-q6cr74, klula-q6cr90, klula-q6crs0, klula-q6crv8, klula-q6crz9, klula-q6cst8, klula-q6csv8, klula-q6ctp8, klula-q6cu02, klula-q6cu78, klula-q6cu79, klula-q6cuv3, klula-q6cvd3, klula-q6cw70, klula-q6cw92, klula-q6cwu7, klula-q6cx84, klula-q6cxa3, klula-q6cy41, yarli-apth1, yarli-atg15, yarli-BST1B, yarli-lip2, yarli-LIP3, yarli-LIP4, yarli-LIP5, yarli-LIP7, yarli-LIP8, yarli-lipa1, yarli-ppme1, yarli-q6bzp1, yarli-q6bzv7, yarli-q6c1f5, yarli-q6c1f7, yarli-q6c1r3, yarli-q6c2z2, yarli-q6c3h1, yarli-q6c3i6, yarli-q6c3l1, yarli-q6c3u6, yarli-q6c4h8, yarli-q6c5j1, yarli-q6c5m4, yarli-q6c6m4, yarli-q6c6p7, yarli-q6c6v2, yarli-q6c7h3, yarli-q6c7i7, yarli-q6c7j5, yarli-q6c7y6, yarli-q6c8m4, yarli-q6c8q4, yarli-q6c8u4, yarli-q6c8y2, yarli-q6c9r0, yarli-q6c9r1, yarli-q6c9u0, yarli-q6c9v4, yarli-q6c209, yarli-q6c225, yarli-q6c493, yarli-q6c598, yarli-q6c687, yarli-q6c822, yarli-q6cau6, yarli-q6cax2, yarli-q6caz1, yarli-q6cb63, yarli-q6cba7, yarli-q6cbb1, yarli-q6cbe6, yarli-q6cby1, yarli-q6ccr0, yarli-q6cdg1, yarli-q6cdi6, yarli-q6cdv9, yarli-q6ce37, yarli-q6ceg0, yarli-q6cep3, yarli-q6cey5, yarli-q6cf60, yarli-q6cfp3, yarli-q6cfx2, yarli-q6cg13, yarli-q6cg27, yarli-q6cgj3, yarli-q6chb8, yarli-q6ci59, yarli-q6c748, canga-q6fpj0, klula-q6cp11, yarli-q6c4p0, debha-q6btp5, debha-kex1

Abstract

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

ESTHER: Pignede_2000_J.Bacteriol_182_2802
PubMedSearch: Pignede 2000 J.Bacteriol 182 2802
PubMedID: 10781549
Gene_locus related to this paper: aspnc-a5abh9, yarli-lip2

Abstract

We isolated the LIP2 gene from the lipolytic yeast Yarrowia lipolytica. It was found to encode a 334-amino-acid precursor protein. The secreted lipase is a 301-amino-acid glycosylated polypeptide which is a member of the triacylglycerol hydrolase family (EC 3.1.1.3). The Lip2p precursor protein is processed by the KEX2-like endoprotease encoded by XPR6. Deletion of the XPR6 gene resulted in the secretion of an active but less stable proenzyme. Thus, the pro region does not inhibit lipase secretion and activity. However, it does play an essential role in the production of a stable enzyme. Processing was found to be correct in LIP2(A) (multiple LIP2 copy integrant)-overexpressing strains, which secreted 100 times more activity than the wild type, demonstrating that XPR6 maturation was not limiting. No extracellular lipase activity was detected with the lip2 knockout (KO) strain, strongly suggesting that extracellular lipase activity results from expression of the LIP2 gene. Nevertheless, the lip2 KO strain is still able to grow on triglycerides, suggesting an alternative pathway for triglyceride utilization in Y. lipolytica..