Orientation of FtsH protease homologs in Trypanosoma brucei inner mitochondrial membrane and its evolutionary implications.

Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.

Structure of a mitochondrial ATP synthase with bound native cardiolipin.

The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 Å resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids.

Unraveling the subtleties of ß-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families.

Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural ß-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on ß-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on ß-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized ß-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on ß-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli We found that PapP acts on ß-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 ß-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding ß-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.

Development of screening strategies for the identification of paramylon-degrading enzymes.

Enzymatic degradation of the ß-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-ß-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis ß-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.

A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions.

Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.

Low Temperature Stress Alters the Expression of Phytoene Desaturase Genes (crtP1 and crtP2) and the ζ-Carotene Desaturase Gene (crtQ) Together with the Cellular Carotenoid Content of Euglena gracilis.

Carotenoids participate in photosynthesis and photoprotection in oxygenic phototrophs. Euglena gracilis, a eukaryotic phytoflagellate, synthesizes several carotenoids: ß-carotene, neoxanthin, diadinoxanthin and diatoxanthin. Temperature is one of the most striking external stimuli altering carotenoid production. In the present study, to elucidate the regulation of carotenoid synthesis of E. gracilis in response to environmental stimuli, we functionally identified phytoene desaturase genes (crtP1 and crtP2) and the ζ-carotene desaturase gene (crtQ) of this alga and analyzed expression of those genes and the composition of major carotenoids in cells grown under cold (20�C) and high-intensity light (HL; 240 �mol photon m-2 s-1) conditions. 20�C-HL treatment increased the transcriptional level of the phytoene synthase gene (crtB), and crtP1 and crtP2, whose products catalyze the early steps of carotenoid biosynthesis in this alga. Cultivation at 20�C under illumination at 55 �mol photon m-2 s-1 (low-intensity light; LL) decreased the cell concentration, Chl and total major carotenoid content by 61, 75 and 50%, respectively, relative to control (25�C-LL) cells. When grown at 20�C-HL, the cells showed a greater decrease in cell concentration and photosynthetic pigment contents than those in 20�C-LL. ß-Carotene, neoxanthin and diadinoxanthin contents were decreased by more than half in 20�C-LL and 20�C-HL treatments. On the other hand, when subjected to 20�C-LL and 20�C-HL, the cells retained a diatoxanthin content comparable with control cells. Our findings suggested that diatoxanthin plays crucial roles in the acclimation to cold and intense light condition. To the best of our knowledge, this is the first report on a photosynthetic organism possessing dual crtP genes.

Physiological role of ß-carotene monohydroxylase (CYP97H1) in carotenoid biosynthesis in Euglena gracilis.

Some carotenoids are found in the Euglena gracilis, including ß-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated ß-carotene to ß-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of ß-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be ß-carotene monohydroxylase, but not ß-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase.

Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.

Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis.

KEY MESSAGE: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.

Continuous Laminaribiose Production Using an Immobilized Bienzymatic System in a Packed Bed Reactor.

The first continuous production system of laminaribiose from sucrose and glucose in a bienzymatic reaction is reported in this study. Immobilized laminaribiose phosphorylase and sucrose phosphorylase were used in a packed bed reactor system comprising of a 3-cm glass column at 35 °C with a steady feeding flow rate of 0.1 ml/min. Factors affecting product formation including enzyme ratio, peal concept (both enzymes in one pearl or in separate pearls), and pearl size were studied. An enzyme ratio of 2:1 of laminaribiose phosphorylase (LP) to sucrose phosphorylase (SP) when encapsulated separately in bigger size peals resulted in higher concentration of product. Laminaribiose (0.4 g/(L h)) is produced in the optimized system at steady state. The reaction system proved to be operationally stable throughout 10 days of continuous processing. A half-life time of more than 9 days was observed for both biocatalysts.

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain.

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)4-6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)2 and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)2 or GlcNAc, which are easily taken up by the cells.

Identification of Euglena gracilis ß-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149.

Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. ß-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a ß-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a ß-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial ß-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.

Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis.

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.

Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis.

BACKGROUND: Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. RESULTS: After cultivation for 7 days under continuous light at 920 µmol m-2 s-1, ß-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 µmol m-2 s-1). The high-intensity light (920 µmol m-2 s-1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress. CONCLUSIONS: Our results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress.

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions.

In Euglena gracilis, pyruvate:NADP+ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP+ oxidoreductase. In contrast, pyruvate:NADP+ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.

Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis.

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, ß, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αß)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and ß subunits to the fundamental process of ATP synthesis.

Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes.

Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and ß-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.

Immobilization and Characterization of E. gracilis Extract with Enriched Laminaribiose Phosphorylase Activity for Bienzymatic Production of Laminaribiose.

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).

Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

BACKGROUND: Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains ß-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. RESULTS: To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 µmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 µmol m(-2) s(-1)). When grown under continuous light at 920 µmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. CONCLUSIONS: We identified genes encoding CrtE and CrtB in E. gracilis and found that their protein products catalyze the early steps of carotenoid biosynthesis. Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena.