Oleic Acid Medium Research Articles

Abstract 3068: CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming

Abstract Background & Aim: In the tumor microenvironment, the adipocytes enhance the invasiveness of breast cancer (BC) by remodeling the lipid metabolism, especially in triple-negative breast cancer (TNBC). The cell adhesion molecule CD146 was highly expressed in TNBC and predicted poor distant metastasis free survival (DMFS) in patients. Meanwhile, CD146 was known to alter the obesity process by regulating the metabolic level of the body. In this study, we aimed to explore the molecular mechanism of CD146 in tumor metastasis mediating by lipid metabolic reprogramming between adipocytes and TNBC cells. Methods: CD146 knockout (KO) or overexpression cell models were constructed by CRISPR/Cas9 or lentivirus. Cells labeled with luciferase were injected in tail vein of Balb/c-nude mice and monitored lung metastasis by IVIS Kinetic system in vivo. Transwell and wound healing assays were analyzed cell aggressiveness in vitro. We synthesized anti-CD146 nanobodies, which significantly degraded CD146 protein and inhibitd its function. RNA-seq and Lipidomics in CD146 knockout models were performed to analyze the role of CD146 in TNBC cell metabolism. Immunofluorescence was used to detect the intracellular content of Lipid Droplet (LD) and protein localization. Finally, we used oleic acid (OA) and adipocyte-conditioned medium (CM) to mimic the tumor microenvironment. Results: CD146 was highly expressed in TNBC and predicted poor DMFS survival. Inhibition of CD146 suppressed tumor aggressiveness and lung metastasis in vitro and in vivo. According to the bioinformatics and RNA-Seq analysis, we showed that CD146 was intimately involved in the lipid metabolic pathway, particularly fatty acid metabolism, and revealed that inhibiting CD146 in TNBC elevated the intracellular triglyceride level through lipidomic assay. Meanwhile, knockout of CD146 promoted LD accumulation and decreased fatty acid oxidation (FAO) and mitochondrial ATP levels. In molecular mechanism, we found that adipocytes secrete high level of IL6 and fatty acid, which activated JAK/STAT3 signaling and FAO mediating by CD146 in TNBC cells. Blocking CD146 using its nanobody inhibited IL6/JAK/STAT3 activities and ACADM, leading to impairment of FAO and lung metastasis suppression in vitro and in vivo. Conclusions: In this study, we demonstrate the crucial role of CD146 in the crosstalk between tumor cells and adipocytes in the tumor microenvironment. CD146 promotes the utilization of fatty acids through fatty acid oxidation and activates the downstream inflammatory pathway response with adipocyte-secreted IL-6, thereby driving BC aggressiveness. Our study has important implications for the development of TNBC therapeutics to block CD146-driven FAO process, inflammatory response and tumor metastasis. Citation Format: Hexing Sun, Kaiyuan Huang, Bingmei Sun, Jiajia Lin, Ruiqian Yang, Jiawei Li, Shimeng Wang, De Zeng, Haoyu Lin, Yuanke Liang. CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3068.

Microwave-assisted synthesis of oleic acid-capped silver nanoparticles as lubricant additives

Recently, metal nanoparticles have been studied extensively as lubricant additives. Owning to their nanometer size, the nanoparticles can fill micro defects on contacting surfaces, acting as repairing agents. In this work, we describe a simple and fast method to fabricate monodisperse silver nanoparticles using oleic acid as a capping agent. In particular, silver nitrate was reduced by oleylamine in the oleic acid medium using acetonitrile as a co-solvent, and the reaction was heated by a microwave source. Results showed that the particle size was greatly affected by varying the reductant concentration. The average diameter of synthesized nanoparticles ranges from 3.0 nm to 4.0 nm at optimum conditions. The dispersibility in the oil of the product is attributed to the long-chain alkyl from fatty acid grafted on the surface layer, which constitutes about 21% of the weight of the nanoparticles as determined by thermogravimetric analysis.

Increased peroxisome proliferation is associated with early yeast replicative ageing.

Peroxisomes are single membrane-bound organelles ubiquitously present in several cell types and are associated with cell and tissue-specific functions. Their role in cellular ageing is under investigation in various model systems. Metabolism of cellular reactive oxygen species is a universal function performed by these organelles. In this study, we investigated alterations in peroxisome number upon early replicative ageing of yeast cells. Increase in the number of peroxisomes in replicatively aged mother cells of wild-type yeast was observed when cultured in both peroxisome-inducing and non-inducing medium. Further, we investigated if this increase in peroxisome number in replicatively aged cells is due to enhanced peroxisome proliferation. For this, the number of peroxisomes in replicatively aged mother cells of pex11, pex25 and pex11pex25 was analysed. Increased percentage of aged cells was observed in pex25 and pex11pex25 cells cultured in peroxisome-inducing oleic acid medium. Interestingly, when cultured in oleic acid, young mother cells devoid of Pex11 showed reduced peroxisome proliferation compared to old mother cells. Induced activity of the antioxidant enzyme catalase and reduced accumulation of reactive oxygen species were reported in all studied strains when cultured in oleic acid medium. Further, our data also suggest that replicatively aged cells with increased peroxisome number also display mitochondrial dysfunction and fragmentation in all the strains studied. In conclusion, our data suggests a correlation between increase in peroxisome number and replicative age of yeast cells and interestingly this increase seems to be partly dependent on the fission proteins.

Hydroxytyrosol Attenuates Hepatic Fat Accumulation via Activating Mitochondrial Biogenesis and Autophagy through the AMPK Pathway.

Two experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in Megalobrama amblycephala. Triplicate groups of fish were fed four test diets: (1) low-fat diet (LFD, 5% fat), (2) high-fat diet (HFD, 15% fat), (3) LFD + 100 mg/kg HT (LFD + HT), and (4) HFD + 100 mg/kg HT (HFD + HT) (in vivo). Hepatocytes from the same batch were exposed to three media including L-15 medium (L15), oleic acid (OA) medium [L15 + 400 μM OA], and OA + HT medium [L15 + 400 μM OA + 10 μM HT] to explore the roles of HT in mitochondrial function (in vitro). Fish fed HFD had excessive fat deposition in the liver, and HT inclusion in the HFD decreased hepatic fat deposition. Transmission electron microscopy revealed that the HFD triggers loss of cristae and metrical density and hydropic changes in mitochondria and that HT supplementation attenuates the ultrastructural alterations of mitochondria. The in vitro test showed that HT decreases fat deposition in hepatocytes, suppresses the reactive oxygen species formation, and facilitates the expression of phospho-AMPK protein and the genes involved in mitochondria biogenesis (PGC-1, NRF-1, TFAM) and autophagy (PINK1, Mul1, Atg5). These findings suggest the lipid-lowering effect of HT mediated by activation of mitochondrial biogenesis and autophagy through the AMPK pathway.

Vac8 Controls Vacuolar Membrane Dynamics during Different Autophagy Pathways in Saccharomyces cerevisiae.

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8Δ strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.

Nitrogen-doped carbon quantum dots as fluorescent probe for “off-on” detection of mercury ions, l-cysteine and iodide ions

The application of fluorescent nanoparticles to the detection of inorganic ions and organic compounds has been attracted wide attention recently. In this paper, an “off-on” method for highly sensitive and selective detection of Hg2+ and l-cysteine (l-Cys) or Hg2+ and I− using home-made nitrogen-doped carbon quantum dots (N-CQDs) as fluorescent probe was reported. The N-CQDs with a fluorescence quantum yield of 42.2% were prepared using tartaric acid, citric acid and ethanediamine as the precursors in the oleic acid media. The fluorescence of the obtained N-CQDs could be quenched selectively and sensitively by the addition of Hg2+ (turn-off) with a detection limit of 83.5nM. When l-Cys or I− was added into the N-CQDs-Hg2+ system, the fluorescence was recovered effectively (turn-on). This process could be used to the detection of l-Cys or I− with a detection limit of 45.8 and 92.3nM, respectively.

Synthesis of γ-Fe2O3 Nanoparticles Capped with Oleic Acid and their Magnetic Characterization

In recent years, superparamagnetic iron oxide nanoparticles have attracted a great attention due to their various biomedical applications, such as magnetic resonance imaging, targeted drug delivery, and hyperthermia. In this article, γ-Fe2O3 magnetic nanoparticles (Maghemite) were prepared in oleic acid media by co-precipitation method. The oleic acid, a monounsaturated fatty acid was used as the capping and stabilizing agent during the synthesis of the magnetic nanoparticles. Characterization of obtained nanoparticles were performed using powder X-ray diffraction (PXRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectra (FTIR), and vibrating sample magnetometer (VSM). The crystallite size of γ-Fe2O3 nanoparticles was achieved in the range between 16.2 and 26.8 nm. The FESEM demonstrated the regular spheres of γ-Fe2O3 nanoparticles. The obtained nanoparticles were coated with oleic acid indicating by FTIR analysis. The resulted oleic acid-coated nanoparticles were shown superparamagnetic properties (~52 emu/g). This suggested method is simple and rapid to fabricate superparamagnetic nanoparticles which make them appropriate candidates for theranostic application in future studies.

Oleic acid, hydroxytyrosol and n-3 fatty acids collectively modulate colitis through reduction of oxidative stress and IL-8 synthesis; in vitro and in vivo studies

Our recent study has demonstrated that medium chain triglycerides (MCT) and monounsaturated fatty acids potentiate the beneficial effects of fish oil on risk factors of cardiovascular disease. In the present study, we have investigated the influence of MCT or olive oil on the protective and mucosal healing ability of fish oil in ulcerative colitis using cell simulation and animal models. Caco-2 cells grown in medium chain fatty acids enriched medium has exaggerated t-butyl hydroperoxide induced cell damage, GSH depletion, and IL-1β induced IL-8 synthesis, compared to the cells grown in oleic acid & hydroxytyrosol (OT) enriched medium. Further, combined treatment of cells with eicosapentaenoic acid, docosahexaenoic acid, and OT has remarkably attenuated the cell damage, and IL-8 synthesis, compared to individual treatments. To evaluate the effect of these lipid formulations in vivo, adult Wistar rats were fed diet enriched with high amount of medium chain triglycerides (MCT), virgin olive oil, or their combination with fish oil. Colitis was induced in rats using dextran sulfate sodium (DSS) for 7days followed by 10-days of recovery period. Rats of MCT group exhibit severe disease activity, higher levels of inflammatory cytokines in the colon compared to the olive oil group. Furthermore, there was persistent body weight loss, loose stools, higher levels of inflammatory cytokines in the rats of MCT group, even after DSS was withdrawn from drinking water. Conversely, fish oil has remarkably attenuated the DSS induced alterations in both MCT and olive oil diet groups with significantly greater effect in the olive oil group. Thus, MCT increase the susceptibility to colitis through oxidative damage and IL-8 synthesis in intestinal epithelial cells. The beneficial effects of virgin olive oil could be partially attributed to hydroxytyrosol. Combined treatment of hydroxytyrosol, oleic acid and n-3 fatty acids exhibit huge therapeutic benefits in colitis.

Fabrication of nano-sized copper powders in liquid media via high-energy electrical explosion method: Use of high purity copper recovered from waste jelly-filled cable as raw material

Abstract A novel process for fabrication of nano-sized copper powders in organic oleic acid medium was developed employing a high-energy electrical explosion method. Formation of copper oxides was inhibited by the oleic acid medium during the electrical explosion of copper wires recovered from waste jelly-filled cable as a raw material for synthesis of nano-colloids and final dried nano-sized copper powders. High voltage of 320 V was repeatedly applied between the two horizontal electrodes for 3000 electrical explosions across the horizontal electrodes assembly. The copper powders were finally recovered via dispersion of the colloid by ultra-sonification and subsequent drying in an oven at 60 °C for 1 h and 5 h, respectively. The copper powders thus fabricated were analyzed using FESEM, BET surface area analyzer, TEM and elemental analyzer for determination of morphology, porosity and elemental purity. All of them turned out to be superior compared to similarly produced copper nano-sized powders in deionized water or ethyl alcohol (EtOH) media.

ZnSe Quantum Dots through a Facile one Pot Synthesis Process

A novel one pot synthesis approach in oleic acid medium was employed to obtain monophasic ZnSe quantum dots (QD) of average size 3.7nm. The QDs were well crystalline in hexagonal phase as revealed by x-ray diffraction and high resolution transmission electron microscopy (HRTEM) studies. The ZnSe QDs exhibit sharp emission peak in the blue (465nm) with 385picosecond fluorescence decay time. The theoretical band gap corresponding to 3.7nm ZnSe QDs matched well with the measured 3.11eV band gap of synthesized QDs which thus showed quantum confinement effect.

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica.

Peroxisomes are essential organelles in the cells of most eukaryotes, from yeasts to mammals. Their role in β-oxidation is particularly essential in yeasts; for example, in Saccharomyces cerevisiae, fatty acid oxidation takes place solely in peroxisomes. In this species, peroxisome biogenesis occurs when lipids are present in the culture medium, and it involves the Pex11p protein family: ScPex11p, ScPex25p, ScPex27p, and ScPex34p. Yarrowia lipolytica has three Pex11p homologues, which are YALI0C04092p (YlPex11p), YALI0C04565p (YlPex11C), and YALI0D25498p (Pex11/25p). We found that these genes are regulated by oleic acid, and as has been observed in other organisms, YlPEX11 deletion generated giant peroxisomes when mutant yeast were grown in oleic acid medium. Moreover, ΔYlpex11 was unable to grow on fatty acid medium and showed extreme dose-dependent sensitivity to oleic acid. Indeed, when the strain was grown in minimum medium with 0.5% glucose and 3% oleic acid, lipid body lysis and cell death were observed. Cell death and lipid body lysis may be partially explained by an imbalance in the expression of the genes involved in lipid storage, namely, DGA1, DGA2, and LRO1, as well as that of TGL4, which is involved in lipid remobilization. TGL4 deletion and DGA2 overexpression resulted in decreased oleic acid sensitivity and delayed cell death of ΔYlpex11, which probably stemmed from the release of free fatty acids into the cytoplasm. All these results show that YlPex11p plays an important role in lipid homeostasis in Y. lipolytica.

Immobilizing heteropolyacids on zirconia-modified silica as catalysts for oleochemistry transesterification and esterification reactions

A new method of chemical immobilization of Keggin heteropolyacids (HPAs) was suggested. H3PW12O40, H4SiW12O40, and H3PMo12O40 were immobilized on the silica which was previously grafted with zirconium butoxide. The immobilization method promoted strong interaction HPA-support and yielded 25wt.% of well-dispersed HPAs, so increasing the density of acid sites. The catalysts were active in the reaction of transesterification of methyl stearate with n-butanol and esterification of oleic acid with trimethylolpropane. We demonstrate that, contrary to the immobilized H3PMo12O40, the H3PW12O40 and H4SiW12O40-based catalysts are stable toward leaching in a non-polar oleic acid medium. A discussion on circumventing the leaching in non-polar versus polar media is proposed in terms of interaction strength HPA-support. The stronger interaction (i.e., better resistance for leaching) between the support and H3PW12O40 (or H4SiW12O40) is referred to the lower difference of electronegativity between Zr and W and the lower polarizability of the bonds Zr–O–W compared to Zr–O–Mo.

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H2O2 is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1∆ cells, and subsequently, the growth of gpx1∆ cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1C36S was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.

Electrosynthesis and catalytic properties of silver nano/microparticles with different morphologies

Electrosynthesis of powdery silver particles can be effectively carried out with an H 2O–oleic acid or an H 2O–glycerol mix solvent (volume ratio 1:1) as the electrolytic medium and AgNO 3 as the supporting electrolyte. Experimental results indicate that the presence or absence of the surfactant sodium dodecyl sulfate (SDS) and the choice of electrolytic medium have a significant impact on the shape and size of the prepared Ag particles. With H 2O–glycerol as the electrolytic medium, spherical Ag nanoparticles can be obtained in the presence of SDS (0.6 g/L), while an Ag sample electrodeposited without SDS has a dendritic microcrystalline structure. For the reduction of methyl orange (MO) and methylene blue (MB) with NaBH 4 as the reducing agent, the spherical Ag nanoparticles exhibit much better catalytic activity than the dendritic Ag microparticles. Further investigations show that surface modification by an oleic acid medium could greatly improve the catalytic activity of the electrodeposited Ag particles for the reduction of MO and MB.

SOAgenes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeastYarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene (LIP2) was monitored using a LIP2-beta-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.

Fluorescence quenching of CdSe quantum dots by nitroaromatic explosives and their relative compounds

CdSe quantum dots (QDs) were synthesized in oleic acid and octadecene medium under high-temperature and dispersed in chloroform. Nitroaromatic explosives and their relative compounds, 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), nitrobenzene (NB), 2,4-dinitrochlorobenzene (DNBCl) and p-nitrotoluene (NT) can obviously cause the fluorescence quenching of the synthesized QDs. Under the optimum conditions, a nonlinear response was observed over the concentration range of 10 −8 to 10 −5 M for them all. The modified Stern–Volmer quenching equations of ln I 0/ I versus C show a good linear relation in 10 −5 M order of magnitude, and the detection limits approach 10 −6 to 10 −7 M.

Pex19p-dependent Targeting of Pex17p, a Peripheral Component of the Peroxisomal Protein Import Machinery

Pex19p is required for the topogenesis of peroxisomal membrane proteins (PMPs). Here we have demonstrated that Pex19p is also required for the peroxisomal targeting and stability of Pex17p, a peripheral component of the docking complex of the peroxisomal protein import machinery. We have demonstrated that Pex17p is associated with the peroxisomal Pex13p-Pex14p complex as well as with Pex19p. We have identified the corresponding binding sites for Pex14p and Pex19p and demonstrated that a specific loss of the Pex19p interaction resulted in mistargeting of Pex17p. We have shown that a construct consisting only of the Pex19p- and Pex14p-binding sites of Pex17p is sufficient to direct an otherwise cytosolic reporter protein to the peroxisomal membrane in a Pex19p-dependent manner. Our data show that the function of Pex19p as chaperone or import receptor is not restricted to integral membrane proteins but may also include peripheral PMPs. As a consequence of our data, the previous definition of a targeting signal for PMPs (mPTS) as a Pex19p-binding motif in conjunction with a transmembrane segment should be extended to regions comprising a Pex19p-binding motif and a peroxisomal anchor sequence.

Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.

Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.

Acyl-CoA oxidase, a key step for lipid accumulation in the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to use fatty acids as carbon source via β-oxidation. This yeast contains five acyl-CoA oxidases (Aoxps) encoded by POX genes. Two Aoxps are of particular interest: Aox2p, a medium chain oxidase, and Aox3p, an oxidase specific for short chain substrates. To date, no 3D structure of these enzymes is available. To better understand their specificity, we have expressed 10 enzymes in Escherichia coli; Aox2p, Aox3p, a mutated Aox3 (Aox3mp), and seven chimera obtained by shuffling fragments of Aox2p and Aox3p (M1 to M4 and M6 to M8). Aox3mp and one chimeric protein (M6) were active, Aox3mp exhibited broad chain length specificity. We tested complementation of growth for strain MTLY36 (Δpox2,3,5; reduced growth on oleic acid media) and MTLY40 (Δpox2,3,4,5; not growing on oleic acid media) by expressing either Aox2p, Aox3p, Aox3mp, and M6 chimera encoding genes. We demonstrate that the POX genomic content affects both γ-lactone production [J. Mol. Catal. B, this issue] and lipid storage. Strain MTLY36 having Aox4p could still use oleic acid but grew slower than the wild type strain and did not accumulate lipids. The strains containing Aox3p, Aox3mp (alone or with Aox4p) grew on oleic acid and stored lipids as well as did the wild type. Strains expressing Aox2p (MTLY40-2P) and Aox2p, Aox4p (MTL36-2P) stored more lipids than the wild type. Mastering POX genotype allowed us to construct strains, able to accumulate high lipid content, which could be used for single cell oil (SCO) production.

Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica.

To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.