CARACTERISATION BIOCHIMIQUE ET STRUCTURALE D'UNE -GLYCOSIDASE DE THERMUS THERMOPHILUS SUREXPRIMEE CHEZ E. COLI

NANTES-BU Sciences (441092104) / SudocSudocFranceF

Effect of enzyme addition on fermentative hydrogen production from wheat straw

International audienceWheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H-2), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H-2 usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H-2 production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H-2 production from untreated wheat straw ranged from 5.18 to 10.52 mL-H-2 g-VS-1. Whatever the experimental enzyme addition procedure, a two-fold increase in H-2 production yields ranging from 11.06 to 19.63 mL-H-2 g-VS-1 was observed after enzymatic treatment of the wheat straw. The high variability in H-2 yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H-2 production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw(-1)

De l’art dramatique à l’art de l’entrevue médicale : retour d’une expérience auprès d’étudiants en médecine

Contexte : Tant l’enseignement du savoir-faire que celui du savoir-être, en particulier la communication médecin-patient peuvent être améliorés. Objectifs : Décrire et documenter l’intérêt des étudiants pour des dispositifs de formation à la communication et à la relation médecin-patient associant l’art dramatique et la simulation. Méthodes : Trente-trois étudiants en médecine (diplôme de formation approfondie en sciences médicales 1 et 2) ont participé à des ateliers de communication associant successivement une approche théâtrale comportant plusieurs exercices et une mise en situation professionnelle de communication en situation de consultation avec un patient simulé. Résultats : La grande majorité (92 %) des étudiants ayant participé à ces ateliers ont estimé que l’expérience leur a été bénéfique tant sur le plan du savoir-faire que du savoir-être et 94 % ont validé l’intérêt de ces ateliers pour leur pratique ultérieure. Les exercices théâtraux les plus plébiscités étaient l’histoire en cercle, l’histoire à plusieurs et l’histoire avec contrainte. Conclusion : L’association d’outils pédagogiques contextualisants, comme l’expérience théâtrale, le recours aux patients simulés en situation de problématiques cliniques fréquentes ou complexes, à des étudiants observateurs, le recours à la vidéo, et le débriefing entre les différents acteurs, recueille une très forte adhésion et détermine un grand bénéfice ressenti auprès des apprenants. Les résultats très positifs de cette expérience nous encouragent à étendre cette approche pédagogique intégrative à l’ensemble des étudiants du deuxième cycle des études médicales

Binding of the dystrophin second repeat to membrane di-oleyl phospholipids is dependent upon lipid packing.

International audienceDystrophin is the genetically deficient protein in Duchenne Muscular Dystrophy. Its C- and N-terminal ends interact with cytoskeletal and membrane proteins, establishing a link between the cytoskeleton and the extracellular matrix. In a previous study, we showed that there is an interaction between the second repeat of the rod domain and membrane phospholipids, which places tryptophan residues in close contact with the membrane. Here, we examine the binding of the dystrophin repeat-2 to small unilamellar vesicles with varying composition. We find that the protein binds predominantly to di-oleyl-phosphatidylserine. The binding as a function of increasing mol% of DOPS appears to be cooperative due to reduction of dimensionality, greatly enhanced in the absence of salts, and partly modulated by pH. Substituting small by large unilamellar vesicles induces a 30-fold lower affinity of the protein for the membrane phospholipids. However, modifying the packing of the acyl chains by introducing lipids such as phosphatidylethanolamine and cholesterol to the vesicle leads to an approximately 7-fold increase in affinity. Taken together, these results show that the binding involves electrostatic forces in addition to hydrophobic ones

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

International audienceThe metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane en-velope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) producedin the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids viavarious routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of theBubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for theproduction of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBGpoint mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specificincrease of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacyl-glyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structuralmodeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defectivein acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports acritical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess satu-rated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content inthis microalg

MOESM3 of In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

Additional file 3: Table S1. Primers used for cloning and site-directed mutagenesis

MOESM1 of In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

Additional file 1: Figure S1. Protein sequence alignment of CYP-Aa162 from A. acidocaldarius (GenBank Accession Number: WP_008340313), P450BSβ from Bacillus subtilis str. 168 (GenBank Accession Number: NP_388092), OleTJE from Jeotgalicoccus sp. ATCC 8456 (GenBank Accession Number: ADW41779), CYP-Sm46 (labelled as Sm46 extended) from Staphylococcus massiliensis S46 (GenBank Accession Number: EKU50422), CYP-Sm46Δ29 (labelled as Sm46Δ29) from S. massiliensis (GenBank Accession Number: WP_039990689) and cytochrome P450 enzymes from other Staphylococcus species such as S. agnetis (GenBank Accession Number: KFE42911), S. delphini (GenBank Accession Number: WP_019165531), S. intermedius (GenBank Accession Number: WP_019167377) and S. pseudintermedius HKU10-03 (GenBank Accession Number: ADV05454). Figure S2. SDS-PAGE showing the purified His-tagged CYP-Aa162 (lane A) and CYP-Sm46Δ29 (lane S). Molecular sizes of the marker bands (lane M), from top to bottom, are 180, 135, 100, 75, 63, 48, 35 and 25 kDa respectively. Figure S3. The UV–visible spectra of CYP-Sm46Δ29 (5 μM) under different conditions. (A) The purified CYP-Sm46Δ29 was diluted in 50 mM Na3PO4 (pH 7.4) buffer containing 300 mM NaCl and 10% glycerol. Spectra are shown for the oxidized ferric form of the enzyme (orange line) and the ferrous-CO complex reduced by the indicated amount of Na2S2O4. (B) The purified CYP-Sm46Δ29 was diluted in 50 mM Na3PO4 buffer containing 300 mM NaCl and 10% glycerol with different buffer pH as indicated. Then the absorption spectra were recorded respectively for the oxidized ferric form and the ferrous-CO adduct reduced by 10 mM Na2S2O4. The protein precipitates at buffer pH lower than 7.0. (C) A molar excess (600 μM) of C12 lauric acid was pre-incubated with the enzyme at room temperature for 5 min before the absorption spectra were recorded. Binding of C12 FA did not seem to induce an apparent spin-state transition of the ferric heme. The Soret peak of the C12-bound ferrous-CO adduct of the enzyme was still detected at 420 nm. Figure S4. UV–visible spectra of the self-sufficient monooxygenase P450BM3. The substrate-bound ferric form of P450BM3 (solid line) shows a Soret maximum at ~ 416 nm with undistinguishable β-band and a weaker α-band at 570 nm. The reduced ferrous-CO form of P450BM3 (dashed line) generated by the subsequent NADPH-initiated electron transfer features a shifted Soret peak to 448 nm. Figure S5. Effect of decoupling NADH oxidation and electron transfer on the catalytic conversion of lauric acid (LA) by CYP-Aa162 and CYP-Sm46Δ29. The reactions contained 0.2 mM LA, 2.0 μM CYP-Aa162 (or CYP-Sm46Δ29), 3.0 μM putidaredoxin reductase (PdR), 1 mM NADH in the absence and presence of 1200 U mL−1 catalase (Catl.). By subtracting putidaredoxin (Pdx) from the reaction system, the NADH oxidation was mandatorily decoupled from the Class I electron transfer chain to P450 enzymes. Any catalytic activity observed should be supported by the H2O2 generated from NADH oxidation and O2 reduction. The percentage conversion of LA was determined by calculating the substrate consumption based on GC analysis. Results shown are mean ± SD of duplicated experiments. Figure S6. The ratios of free fatty acid (FFA) decarboxylation (DC) over hydroxylation (HD) by CYP-Sm46Δ29 against different FFA substrates. The decarboxylation activity was measured by detecting the 1-alkene yield using GC analytical method. The hydroxylation activity was estimated by subtracting the alkene production from the total substrate conversion. This indirect but more convenient method was validated with C14 myristic acid substrate by direct measurement of the BSTFA/TMCS derivatized hydroxylation products. Results are shown as mean ± SD of duplicated experiments. Figure S7. Phylogenetic tree for CYP-Aa162, CYP-Sm46Δ29 and other CYP152 family members. The sequences were aligned using ClustalW. The Neighbor-joining Tree was generated using MEGA 7.0 package. Bootstrap values shown next to the branches were computed from 1000 bootstrap tests. CYP-Sm46 was found most closely related to the P450 fatty acid decarboxylase OleTJE (CYP152L1), while CYP-Aa162 (CYP152A8) is much closer to the P450 fatty acid hydroxylase P450BSβ (CYP152A1). Figure S8. Protein sequence alignment of OleTJE, CYP-Sm46Δ29, CYP-Aa162 and P450BSβ. *: the only two residues that are distinct in the active sites of these four P450 peroxygenases, which are proposed to be important for product distribution; #: the key catalytic residue. Figure S9. Kinetic curves of CYP-Aa162 and CYP-Sm46Δ29 against their optimal fatty acid substrates. (A) C12 lauric acid substrate consumption rates by CYP-Aa162 were fitted to Hill equation; (B) 1-undecene formation rates by CYP-Sm46Δ29 were fitted to Hill equation; (C) Solid line: the plot of 1-tridecene formation rates by CYP-Sm46Δ29 as a function of increasing C14 myristic acid concentrations, demonstrating substantial substrate inhibition. Dotted line: a hyperbolic curve fitted with Michaelis–Menten equation after truncating the inhibited rates at high C14 substrate concentrations. The steady state kinetic parameters were calculated using OriginPro 8.0 and are summarized in Table 2

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids

International audienceNitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response