Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts

Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species. Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid. The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with α-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products

Etude de l'activité galactolipase des lipases pancréatiques apparentées de type 2 (PLRP2)

La famille des lipases pancréatiques est constituée de trois sous-familles : la lipase pancréatique classique (PL) et les lipases pancréatiques apparentées de type 1 et 2 (PLRP1 et PLRP2). Contrairement à la PL, qui est active seulement sur les triglycérides, la PLRP2 possède trois types d’activités enzymatiques : lipase, phospholipase de type A1 et surtout galactolipase. Cette dernière activité est particulièrement intéressante car les galactolipides, principalement les monogalactosyldiacylglycérols (MGDG) et les digalactosyldiacylglycérols (DGDG) sont les lipides les plus abondants dans la nature. Un test continu, spécifique et fiable de dosage de l’activité galactolipase à été mis au point en utilisant la technique du pH-stat et un galactolipide synthétique à chaîne moyenne comme substrat, le MGDG en C8. Ce nouveau test de dosage nous a permis de mesurer pour la première fois des activités spécifiques importantes avec les PLRP2 humaine (HPLRP2) et du cobaye (GPLRP2) (1786±100 et 5420±85 U/mg, respectivement). Nous avons par la suite utilisé cette même technique pour étudier l’hydrolyse par les PLRP2 des galactolipides naturels à chaînes longues purifiés à partir de feuilles d’épinard. Les activités spécifiques mesurées sont du même ordre de grandeur que celles mesurées avec le MGDG en C8 mais aussi le DGDG en C8 synthétique, montrant ainsi que l’activité galactolipase n’est affectée ni par la longueur de la chaîne acyle ni par la présence d’un ou de deux résidus galactoses. Grâce à ce test nous avons pu réaliser un criblage de lipases microbiennes de différentes origines à la recherche d’activités galactolipase. Dans une deuxième partie, nous avons étudié les propriétés physico-chimiques du bis-monoacylglycérol-phopshate (BMP). Nous avons montré que non seulement ce phospholipide rare est hydrolysé par la PLRP2, mais le BMP et la PLRP2 humaine peuvent être trouvés dans la même cellule, le monocyte, suggérant un nouveau rôle physiologique pour la PLRP2.The pancreatic lipase family consists of three sub-families : the classical pancreatic lipase (PL) and the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). On the contrary to PL, which is active only on triglycerides, the PLRP2 possesses three types of enzymatic activities: lipase activity, phospholipase A1 activity and especially galactolipase activity. This latter activity is particularly interesting because monogalactosyl-diacylglycerol (MGDG) and digalactosyldiacyl-glycerol (DGDG) are the most abundant lipids in nature. A specific, continuous and robust galactolipase assay using the pH-stat technique and a synthetic medium chain MGDG as substrate was developed, and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related proteins 2 were tested as model enzymes. This new assay allowed us to measure for the first time high specific activities with both PLRP2 (1786±100 and 5420±85 U/mg, respectively). We then used this technique to study the hydrolysis by PLRP2 of natural long chain galactolipids purified from spinach leaves. The length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain and long chain galactolipids. We also used the pH-stat technique and medium chain MGDG and DGDG for screening several microbial lipases for their galactolipase activity.In a separate study, we investigated the physico-chemical properties of bis-monoacylglycorol-phopshate (BMP) and found that this rare phospholipid was hydrolyzed by PLRP2. Moreover, BMP and human PLRP2 could be found in the same cell, the monocyte, suggesting a new physiological role for PLRP2

Variations in gastrointestinal lipases, pH and bile acid levels with food intake, age and diseases: Possible impact on oral lipid-based drug delivery systems

International audienceThe lipids and some surfactants present in oral lipid-based drug delivery systems are potential substrates for the various lipases involved in gastrointestinal (GI) lipolysis. The levels of these enzymes, together with pH and biliairy secretion, are important parameters that condition the fate of lipid-based formulations (LBF) and the dispersion, solubilization and absorption of lipophilic drugs in the GI tract. Since in vitro methods of digestion are now combined with dissolution assays for a better assessment of LBF performance, it is essential to have a basic knowledge on lipase, pH and bile acid (BA) levels in vivo to develop relevant in vitro models. While these parameters and their variations in healthy subjects are today well documented, in vivo data on specific populations (age groups, patients with various diseases, patients with treatment affecting GI tract parameters, …) are scarce and obtaining them from clinical studies is sometimes difficult due to ethical limitations. Here we collected some in vivo data already available on the levels of digestive lipases, gastric and intestinal pH, and BAs at various ages and in patients with exocrine pancreatic insufficiency, a pathological situation that leads to drastic changes in GI tract parameters and impacts pharmacological treatments

Effect of environmental conditions on various enzyme activities and triacylglycerol contents in cultures of the freshwater diatom, Asterionella formosa (Bacillariophyceae)

International audienceA detailed analysis of triacylglycerols (TAGs) contents, fatty acid patterns and key enzyme activities in the freshwater diatom Asterionella formosa was performed under various conditions, including nitrate, iron and silicon limitation (stress conditions), or bicarbonate and phytohormones supplementation (stimulation conditions). Of all the conditions tested, the addition of bicarbonate produced the greatest increase (5-fold) in TAGs contents compared to the control while the biomass increased. The addition of phytohormones also allowed a significant increase in TAGs of about 3-fold while the biomass increased. Silicon, unlike iron and nitrate limitation, also triggered a significant increase in TAGs contents of 3.5-fold but negatively affected the biomass. Analysis of fatty acid profiles showed that the mono-unsaturated C16:1 fatty acid was the most abundant in A. formosa, followed by C16:0, C14:0 and eicosapentaenoic acid (EPA; C20:5 n-3). EPA levels were found to increase under nitrate and iron limitation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) activities differed with growth conditions. Most enzymes were up-regulated in stimulated cells while in the case of stressed cells, the pattern of activities was more variable. Detailed analysis of all enzyme activities showed that the most important enzyme among those tested was GAPDH which could be a good candidate for genetic engineering of high lipid-producing algae. This study provides a better understanding of key enzymes and biochemical pathways involved in lipid accumulation processes in diatoms

Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves

International audienceThe removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential -linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet

Investigating the permeability of almond cell walls to digestive enzymes

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Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices

International audiencePorcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ