Simultaneous high nutritional single cell oil and lipase production by Candida viswanathii.

BACKGROUND: Omega fatty acids are a family of polyunsaturated fats associated with several health benefits. Lipases are enzymes with potential application in several food processes such as flavor and aroma, surfactants and formulations for the dairy and bakery industries. In this study, single cell oil and lipase production by Candida viswanathii CCR8137 were evaluated simultaneously from renewable carbon sources under nitrogen limitation. METHODS: Enzyme and single cell oil were obtained in submerged cultivations supplemented with triolein, tributyrin, corn oil, sunflower oil, canola oil and olive oil. The effects of glucose on lipid accumulation, fatty acid profile, enzyme production and cell morphology were also evaluated. RESULTS: The highest lipid accumulation (44.5%, w/w) was obtained from triolein, whereas olive oil was the best inducer of lipase synthesis (26.8 U/mL). Nitrogen limiting cultivations were a key parameter for an organic source which showed higher lipid accumulation and enzyme production than the tested inorganic nitrogen source. Glucose was a poor inducer of lipase synthesis, though increased values of lipid accumulation were observed from this carbon source with a maximum of 63.1% (w/w). The fatty acid profile of lipids produced by C. viswanathii CCR8137 showed a high content of omega-9 fatty acid (C18:1 n-9). The addition of glucose to the culture media resulted in the synthesis of essential fatty acids: vaccenic, linolenic and eicosadienoic acids. CONCLUSIONS: Therefore, C. viswanathii CCR8137 strain can be considered as an oleaginous yeast able to accumulate high concentrations of intracellular lipids, which are potential additives for food industry applications as well as being able to simultaneously synthesize high yields of lipase.

Effect of Protein Flexibility from Coarse-Grained Elastic Network Parameterizations on the Calculation of Free Energy Profiles of Ligand Binding.

The characterization of the affinity and binding mechanism of specific molecules to a protein active site is scientifically and industrially relevant for many applications. In principle, this information can be obtained using molecular dynamics (MD) simulations by calculating the free energy profile of the process. However, this is a computationally demanding calculation. Currently, coarse-grained (CG) force fields are very well implemented for MD simulations of biomolecular systems. These computationally efficient force fields are a major advantage to the study of large model systems and/or those requiring long simulation times. The Martini model is currently one of the most popular CG force fields for these systems. For the specific case of protein simulations, to correctly maintain the macromolecular three-dimensional structure, the Martini model needs to include an elastic network (EN). In this work, the effect of protein flexibility, as induced by three EN models compatible with the Martini force field, was tested on the calculation of free energy profiles for protein-ligand binding. The EN models used were ElNeDyn, GoMartini, and GEN. The binding of triolein (TOG) and triacetin (TAG) to a lipase protein (thermomyces lanuginosa lipase-TLL) was used as a case study. The results show that inclusion of greater flexibility in the CG parameterization of proteins is of high importance in the calculation of the free energy profiles of protein-ligand systems. However, care must be taken in order to avoid unjustified large protein deformations. In addition, due to molecular flexibility there may be no absolute need for the center of the ligand to reach the center of the protein-binding site. The calculation of the energy profile to a distance of about 0.5 nm from the active site center can be sufficient to differentiate the affinity of different ligands to a protein.

Integrated selection and identification of bacteria from polluted sites for biodegradation of lipids.

Wastewater treatment plants face major social concern towards removal of problematic pollutants such as fat oils and grease (FOG). In this context, the main objective of the present work was to select natural bacterial isolates from different polluted sites and evaluate them comparatively to isolates from commercial products, for improved bioremediation strategies and bioaugmentation. In total, 196 isolates were analysed for genomic diversity by two PCR-fingerprinting methods and screened for biodegradation potential with pollutants as sole carbon source. The net area under curve (NAUC) was used for preliminary evaluation of growth ability in M9 medium supplemented with oleic acid and triolein. A principal component analysis of all NAUC data showed that natural isolates presented higher overall biodegradation ability and enabled the selection of 11 natural isolates for lipid degradation assays. Selected isolates were identified by 16S rRNA gene sequencing as members of genera with previously described degradative strains, namely, Acinetobacter (1), Aeromonas (2), Bacillus (1), Pseudomonas (1) and Staphylococcus (6). Best biodegradation results in 7-days assay of FOG content removal were 37.9% for oleic acid and 19.1% for triolein by an Aeromonas sp. isolate and a Staphylococcus cohnii isolate, respectively. A respirometry approach confirmed their higher oxygen uptake rates, although longer adaptation phases where required by the Aeromonas sp. isolate. Consequently, these isolates showed great potential for future bioaugmentation products, to promote FOG degradation, for both in situ and ex situ approaches.

Modulation of Higher-order Behaviour in Model Protocell Communities by Artificial Phagocytosis.

Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom-up synthetic biology. The dynamics of a model protocell community is exploited to modulate the function and higher-order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Enzyme-loaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate a range of processes within the host/guest protocells. Specifically, catalase, lipase, or alkaline phosphatase-filled colloidosomes are used to trigger phagocytosis-induced buoyancy, membrane reconstruction, or hydrogelation, respectively, within the MPE droplets. The results highlight the potential for exploiting surface-contact interactions between different membrane-bounded droplets to transfer and co-locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.

Use of Isotope Tracers to Assess Lipid Absorption in Conscious Lymph Fistula Mice.

This protocol provides a comprehensive reference for the evolution of the lymph fistula model, the mechanism of lipid absorption, the detailed procedure for studying lipid absorption using the lymph fistula model, the interpretation of the results, and consideration of the experimental design. The lymph fistula model is an approach to assess the concentration and rate of a range of molecules transported by the lymph by cannulating lymph duct in animals. In this protocol, mice first undergo surgery with the implantation of cannulae in the duodenum and mesenteric lymph duct and are allowed to recover overnight in Bollman restraining cages housed in a temperature-regulated environment. To study in vivo lipid absorption, a lipid emulsion is prepared with labeled tracers, including [3 H]-triolein and [14 C]-cholesterol. On the day of the experiment, mice are continuously infused with lipid emulsion via the duodenum for 6 hr, and lymph is usually collected hourly. At the end of the study, gastrointestinal segments and their luminal contents are collected separately for determination of the digestion, uptake, and transport of exogenous lipids. © 2019 by John Wiley & Sons, Inc.

Role of lipolysis in postoral and oral fat preferences in mice.

Fatty acid receptors in the mouth and gut are implicated in the appetite for fat-rich foods. The role of lipolysis in oral- and postoral-based fat preferences of C57BL/6J mice was investigated by inhibiting lipase enzymes with orlistat. Experiment 1 showed that postoral lipolysis is required: mice learned to prefer (by 70%) a flavored solution paired with intragastric infusions of 5% soybean oil but not a flavor paired with soybean oil + orlistat (4 mg/g fat) infusions. Experiments 2-4 tested the oral attraction to oil in mice given brief choice tests that minimize postoral effects. In experiment 2, the same low orlistat dose did not reduce the strong (83-94%) preference for 2.5 or 5% soybean oil relative to fat-free vehicle in 3-min tests. Mice in experiment 3 given choice tests between two fat emulsions (2% triolein, corn oil, or soybean oil) with or without orlistat at a high dose (250 mg/g fat) preferred triolein (72%) and soybean oil (67%) without orlistat to the oil with orlistat but were indifferent to corn oil with and without orlistat. In experiment 4, mice preferred 2% triolein (62%) or soybean oil (89%) to vehicle when both choices contained orlistat (250 mg/g fat). Fatty acid receptors are thus essential for postoral but not oral-based preferences. Both triglyceride and fatty acid taste receptors may mediate oral fat preferences.

Triglycerides cross the blood-brain barrier and induce central leptin and insulin receptor resistance.

OBJECTIVE: Resistance at the brain receptors for leptin and insulin has been associated with increased feeding, obesity and cognitive impairments. The causal agent for central resistance is unknown but could be derived from the blood. Here we postulate whether hypertriglyceridemia, the major dyslipidemia of the metabolic syndrome, could underlie central leptin and insulin resistance. DESIGN: We used radioactively labeled triglycerides to measure blood-brain barrier (BBB) penetration, western blots to measure receptor activation, and feeding and cognitive tests to assess behavioral endpoints. RESULTS: Human CSF was determined to contain triglycerides, a finding previously unclear. The radioactive triglyceride triolein readily crossed the BBB and centrally administered triolein and peripherally administered lipids induced in vivo leptin and/or insulin resistance at hypothalamic receptors. Central triolein blocked the satiety effect of centrally administered leptin. Decreasing serum triglycerides with gemfibrozil improved both learning and memory inversely proportionate to triglyceride levels. CONCLUSIONS: Triglycerides cross the blood-brain barrier rapidly, are found in human cerebrospinal fluid, and induce central leptin and insulin receptor resistance, decreasing satiety and cognition.

The triolein/aqueous interface and lipase activity studied by spectroscopic ellipsometry and coarse grained simulations.

In spite of the importance of the triglyceride aqueous interface for processes like emulsification, surfactant interactions and lipase activity, relatively little is known about this interface compared to that between alkanes and water. Here, the contact between triolein and water was investigated in terms of water inclusion in the oil phase and orientation of the molecules at the interface. Coarse grained models of triglycerides in contact with water were constructed and correlated with experimental results of the changes in thickness and refractive index, obtained using spectroscopic ellipsometry of spin-coated triolein films. The topography of the layer was revealed by atomic force microscopy. Dry triolein and a triolein sample after equilibration with water were also compared structurally using small-angle X-ray scattering. Additionally, the kinetics of adsorption/activity of three different variants of the Thermomyces lanuginosus lipase (TLL) were investigated. The results show that uptake of water in the triolein phase leads to increase in thickness of the layer. The observed increase of thickness was further enhanced by an active lipase but reduced when an inactive mutant of the enzyme was applied.

Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis.

The alcoholysis of triolein was used to explore the specific features of a recombinant Rhizopus oryzae lipase (rROL) for biodiesel synthesis. For this purpose, different acylglycerols were compared as substrates in lipase-catalysed transesterification. rROL was shown to exhibit a higher specificity towards 1-monoolein than triolein compared to other R. oryzae lipases, being more than 4-fold more specific; in contrast, rROL did not accept 2-monoolein as substrate, concluding that it is highly 1,3-positional specific. Comparing ethanol and methanol as acyl-acceptors, it was observed that the latter caused more lipase inactivation. Regarding alcohols, it was also demonstrated that acyl migration occurred in moderate alcohol concentrations.

Severely impaired activity of lipoprotein lipase Arg243His is partially ameliorated by emulsifying phospholipids in in vitro triolein hydrolysis analysis.

Background We investigated the in vitro effects of various phospholipids as emulsifiers on the hydrolysing activities of lipoprotein lipase (LPL) Arg243His against triolein as substrate. LPL Arg243His, identified in a patient with hyperchylomicronaemia, displays severely diminished activity for triolein when emulsified with Triton X-100. Methods Lipolytic activities of plasma obtained by heparin injection from a homozygous patient with LPL Arg243His were analysed using triolein emulsified with phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), or Triton X-100 as substrates. Results The hydrolysing activities of the patient's plasma for triolein emulsified with PC, PE, PS, PI, LPC and Triton X-100 were 9.22 ± 1.06 µmol/ml/h/ngLPL, 2.94 ± 1.60 µmol/ml/h/ng LPL, 3.72 ± 1.63 µmol/ml/h/ng LPL, 3.40 ± 1.20 µmol/ml/h/ngLPL, 3.72 ± 1.96 µmol/ml/h/ngLPL and 7.80 ± 4.48 µmol/ml/h/ng LPL, respectively. Thus, the specific activities of the patient's LPL determined with triolein emulsified with PC were significantly higher than those with PE, PS, PI or LPC as emulsifiers. Relative to the activities of normal plasma measured with PC, PE, PS, PI and LPC as emulsifiers, the mutant's activities were 49.1 ± 5.2%, 44.1 ± 5.7%, 31.7 ± 12.6%, 19.2 ± 6.9% and 23.8 ± 11.3%, respectively. Using PC, PE, PS, PI and LPC as emulsifiers, the mutant's activities for triolein-lipolysis relative to normal were significantly increased in comparison to the relative activity measured with the classical emulsifier, Triton X-100 (12.9 ± 6.7%). Conclusions Impaired triolein hydrolysis by LPL Arg243His was partially ameliorated by triolein emulsification with phospholipids. The in vitro analysis of triolein hydrolysis using various phospholipid emulsifiers may be useful for the further understanding of impaired LPL function.

Inhaled Vitamin D: A Novel Strategy to Enhance Neonatal Lung Maturation.

INTRODUCTION: The physiologic vitamin D (VD), 1α,25(OH)2D3 (1,25D) is a local paracrine/autocrine effecter of fetal lung maturation. By stimulating alveolar type II cell and lipofibroblast proliferation and differentiation, parenterally administered 1,25D has been shown to enhance neonatal lung maturation; but due to the potential systemic side effects of the parenteral route, the translational value of these findings might be limited. To minimize the possibility of systemic toxicity, we examined the effects of VD on neonatal lung maturation, when delivered directly to lungs via nebulization. METHODS: One-day-old rat pups were administered three different doses of 1,25D and its physiologic precursor 25(OH)D (25D), or the diluent, via nebulization daily for 14 days. Pups were sacrificed for lung, kidneys, and blood collection to determine markers of lung maturation, and serum 25D and calcium levels. RESULTS: Compared to controls, nebulized 25D and 1,25D enhanced lung maturation as evidenced by the increased expression of markers of alveolar epithelial (SP-B, leptin receptor), mesenchymal (PPARγ, C/EBPα), and endothelial (VEGF, FLK-1) differentiation, surfactant phospholipid synthesis, and lung morphology without any significant increases in serum 25D and calcium levels. CONCLUSIONS: Inhaled VD is a potentially safe and effective novel strategy to enhance neonatal lung maturation.

Low-Density Lipoprotein Receptor-Dependent and Low-Density Lipoprotein Receptor-Independent Mechanisms of Cyclosporin A-Induced Dyslipidemia.

OBJECTIVE: Cyclosporin A (CsA) is an immunosuppressant commonly used to prevent organ rejection but is associated with hyperlipidemia and an increased risk of cardiovascular disease. Although studies suggest that CsA-induced hyperlipidemia is mediated by inhibition of low-density lipoprotein receptor (LDLr)-mediated lipoprotein clearance, the data supporting this are inconclusive. We therefore sought to investigate the role of the LDLr in CsA-induced hyperlipidemia by using Ldlr-knockout mice (Ldlr(-/-)). APPROACH AND RESULTS: Ldlr(-/-) and wild-type (wt) C57Bl/6 mice were treated with 20 mg/kg per d CsA for 4 weeks. On a chow diet, CsA caused marked dyslipidemia in Ldlr(-/-) but not in wt mice. Hyperlipidemia was characterized by a prominent increase in plasma very low-density lipoprotein and intermediate-density lipoprotein/LDL with unchanged plasma high-density lipoprotein levels, thus mimicking the dyslipidemic profile observed in humans. Analysis of specific lipid species by liquid chromatography-tandem mass spectrometry suggested a predominant effect of CsA on increased very low-density lipoprotein-IDL/LDL lipoprotein number rather than composition. Mechanistic studies indicated that CsA did not alter hepatic lipoprotein production but did inhibit plasma clearance and hepatic uptake of [(14)C]cholesteryl oleate and glycerol tri[(3)H]oleate-double-labeled very low-density lipoprotein-like particles. Further studies showed that CsA inhibited plasma lipoprotein lipase activity and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. CONCLUSIONS: We demonstrate that CsA does not cause hyperlipidemia via direct effects on the LDLr. Rather, LDLr deficiency plays an important permissive role for CsA-induced hyperlipidemia, which is associated with abnormal lipoprotein clearance, decreased lipoprotein lipase activity, and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Enhancing LDLr and lipoprotein lipase activity and decreasing apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9 levels may therefore provide attractive treatment targets for patients with hyperlipidemia receiving CsA.

WAT apoC-I secretion: role in delayed chylomicron clearance in vivo and ex vivo in WAT in obese subjects.

Reduced white adipose tissue (WAT) LPL activity delays plasma clearance of TG-rich lipoproteins (TRLs). We reported the secretion of apoC-I, an LPL inhibitor, from WAT ex vivo in women. Therefore we hypothesized that WAT-secreted apoC-I associates with reduced WAT LPL activity and TRL clearance. WAT apoC-I secretion averaged 86.9 ± 31.4 pmol/g/4 h and 74.1 ± 36.6 pmol/g/4 h in 28 women and 11 men with BMI ≥27 kg/m(2), respectively, with no sex differences. Following the ingestion of a (13)C-triolein-labeled high-fat meal, subjects with high WAT apoC-I secretion (above median) had delayed postprandial plasma clearance of dietary TRLs, assessed from plasma (13)C-triolein-labeled TGs and apoB48. They also had reduced hydrolysis and storage of synthetic (3)H-triolein-labeled ((3)H)-TRLs in WAT ex vivo (i.e., in situ LPL activity). Adjusting for WAT in situ LPL activity eliminated group differences in chylomicron clearance; while adjusting for plasma apoC-I, (3)H-NEFA uptake by WAT, or body composition did not. apoC-I inhibited in situ LPL activity in adipocytes in both a concentration- and time-dependent manner. There was no change in postprandial WAT apoC-I secretion. WAT apoC-I secretion may inhibit WAT LPL activity and promote delayed chylomicron clearance in overweight and obese subjects. We propose that reducing WAT apoC-I secretion ameliorates postprandial TRL clearance in humans.

Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols.

UNLABELLED: Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. IMPORTANCE: Little is known about how harmless Corynebacterium species that colonize the human nose and skin might impact pathogen colonization and proliferation at these sites. We show that Corynebacterium accolens, a common benign nasal bacterium, modifies its local habitat in vitro as it inhibits growth of Streptococcus pneumoniae by releasing antibacterial free fatty acids from host skin surface triacylglycerols. We further identify the primary C. accolens lipase required for this activity. We postulate a model in which higher numbers of C. accolens cells deter/limit S. pneumoniae nostril colonization, which might partly explain why children without S. pneumoniae colonization have higher levels of nasal Corynebacterium. This work narrows the gap between descriptive studies and the needed in-depth understanding of the molecular mechanisms of microbe-microbe interactions that help shape the human microbiome. It also lays the foundation for future in vivo studies to determine whether habitat modification by C. accolens could be promoted to control pathogen colonization.

Characterization of a new sn-1,3-regioselective triacylglycerol lipase from Malbranchea cinnamomea.

The thermophilic ascomycetous fungus Malbranchea cinnamomea produces lipases (EC 3.1.1.3) that allow it to grow efficiently on medium containing triacylglycerol substrates such as plant oils or tributyrin as sole carbon source. In the transcriptome of M. cinnamomea grown on olive oil, we found one cDNA sequence encoding a putative extracellular lipase. This gene, termed as MclipA, was cloned and heterologously expressed in Pichia pastoris. The recombinant protein, rMclipA, catalyzed the hydrolysis of short-chain fatty acid ester such as p-nitrophenyl butyrate (C4) and long-chain fatty acid ester such as p-nitrophenyl myristate (C14). These results indicate that MclipA is a true triacylglycerol lipase. For rMclipA, the optimum lipase activity was obtained at 45 °C, and more than 93% of enzyme activity was retained after 24 H of incubation at temperatures up to 50 °C. rMclipA was active toward p-nitrophenyl esters of various carbon chain lengths with peak activity on long-chain fatty acid (C14). rMclipA displayed high sn-1,3-regioselectivity on hydrolyzing triolein. rMclipA can catalyze oleic acid methyl ester synthesis resulting in a 71% esterification degree after 24 H of reaction at 40 °C. These properties suggest that rMclipA has potential application in, for example, selective hydrolysis of oil, modification of triacylglycerol, and production of biodiesel.

Study of microwave effects on the lipase-catalyzed hydrolysis.

The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.

Roles of Triolein and Lipolytic Protein in the Pathogenesis and Survival of Mycobacterium tuberculosis: a Novel Therapeutic Approach.

Discovery of novel secreted enzymes and proteins in Mycobacterium tuberculosis (M. tuberculosis) are imperative to understanding the pathogenic system for pathogenesis requires attention. Till date, the groups of these secreted enzymes are not meaningfully characterized in terms of M. tuberculosis. In this way, cutinase, a small lipolytic protein, exists in both bacteria and fungi as well which have a potential catalytic activity. During our search, we have found a few genes of M. tuberculosis demonstrating a same significant lipase action as fungi Fusarium solani cutinase contain. Genome sequencing of M. tuberculosis uncover a lot of proteins, wherein (Rv1758, Rv1984c, Rv2301, Rv3451, Rv3452, Rv3724A, Rv3724B, and Rv3802c) genes have been noted which are exhibiting a cutinase-like activity and closely homologous to that of F. solani cutinase and having the ability to hydrolyze model substrates including p-nitrophenyl butyrate (p-PNB), cutin, triacylglycerols (TAGs), and triolein (TO), yet their biological significance in pathogenesis stays subtle and uncharacterized. In a basic perspective, the measure of cutinase expressed by M. tuberculosis and part of these small lipolytic enzymes in the pathologic discipline require thorough characterization. So, through focusing on cutinase-encoding genes in M. tuberculosis and their active catalytic motif could help to build up a novel therapeutic approach.

Exploring medium-chain-length polyhydroxyalkanoates production in the engineered yeast Yarrowia lipolytica.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or ß-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, we constructed a genetically modified strain of Y. lipolytica by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 with a PTS1 peroxisomal signal. When in single copy, the codon optimized PhaC1 allowed the synthesis of 0.205 % DCW of PHA after 72 h cultivation in YNBD medium containing 0.1 % oleic acid. By using a multi-copy integration strategy, PHA content increased to 2.84 % DCW when the concentration of oleic acid in YNBD was 1.0 %. Furthermore, when the recombinant yeast was grown in the medium containing triolein, PHA accumulated up to 5.0 % DCW with as high as 21.9 g/L DCW, which represented 1.11 g/L in the culture. Our results demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for PHA production using food waste, which contains lipids and other essential nutrients.

Chylomicron remnant model emulsions induce intracellular cholesterol accumulation and cell death due to lysosomal destabilization.

Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization.