Enzyme-aided construction of medium-sized alditols of complete O-linked saccharides The constructed hexasaccharide alditol Galβ1—4GlcNAcβ1—6Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc-ol resists the action of endo-β-galactosidase from Bacteroides fragilis

AbstractWe have constructed by enzyme-aided in vitro synthesis a hexasaccharide alditol Galβ1—4GlcNAcβ1—6Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc-ol and shown that it resists the action of endo-β-galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAcβ1—3Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc-ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo-β-galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAcβ1—6Gal and GlcNAcβ1—3Gal units within linear backbone sequences of all known types of oligo-(N-acetyllactosamino)glycans

New insight on the structural features of the cytotoxic auristatins MMAE and MMAF revealed by combined NMR spectroscopy and quantum chemical modelling

Antibody-drug conjugates (ADCs) are emerging as a promising class of selective drug delivery systems in the battle against cancer and other diseases. The auristatins monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) appear as the cytotoxic drug in almost half of the state-of-the-art ADCs on the market or in late stage clinical trials. Here, we present the first complete NMR spectroscopic characterisation of these challenging molecules, and investigate their structural properties by a combined NMR and quantum chemical modelling approach. We find that in solution, half of the drug molecules are locked in an inactive conformation, severely decreasing their efficiency, and potentially increasing the risk of side-effects. Furthermore, we identify sites susceptible to future modification, in order to potentially improve the performance of these drugs.Peer reviewe

13C-metabolic flux ratio and novel carbon path analyses confirmed that Trichoderma reesei uses primarily the respirative pathway also on the preferred carbon source glucose

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Trichoderma reesei </it>is an important host organism for industrial enzyme production. It is adapted to nutrient poor environments where it is capable of producing large amounts of hydrolytic enzymes. In its natural environment <it>T. reesei </it>is expected to benefit from high energy yield from utilization of respirative metabolic pathway. However, <it>T. reesei </it>lacks metabolic pathway reconstructions and the utilization of the respirative pathway has not been investigated on the level of <it>in vivo </it>fluxes.</p> <p>Results</p> <p>The biosynthetic pathways of amino acids in <it>T. reesei </it>supported by genome-level evidence were reconstructed with computational carbon path analysis. The pathway reconstructions were a prerequisite for analysis of <it>in vivo </it>fluxes. The distribution of <it>in vivo </it>fluxes in both wild type strain and <it>cre1</it>, a key regulator of carbon catabolite repression, deletion strain were quantitatively studied by performing ¹³C-labeling on both repressive carbon source glucose and non-repressive carbon source sorbitol. In addition, the ¹³C-labeling on sorbitol was performed both in the presence and absence of sophorose that induces the expression of cellulase genes. Carbon path analyses and the ¹³C-labeling patterns of proteinogenic amino acids indicated high similarity between biosynthetic pathways of amino acids in <it>T. reesei </it>and yeast <it>Saccharomyces cerevisiae</it>. In contrast to <it>S. cerevisiae</it>, however, mitochondrial rather than cytosolic biosynthesis of Asp was observed under all studied conditions. The relative anaplerotic flux to the TCA cycle was low and thus characteristic to respiratory metabolism in both strains and independent of the carbon source. Only minor differences were observed in the flux distributions of the wild type and <it>cre1 </it>deletion strain. Furthermore, the induction of the hydrolytic gene expression did not show altered flux distributions and did not affect the relative amino acid requirements or relative anabolic and respirative activities of the TCA cycle.</p> <p>Conclusion</p> <p>High similarity between the biosynthetic pathways of amino acids in <it>T. reesei </it>and yeast <it>S. cerevisiae </it>was concluded. <it>In vivo </it>flux distributions confirmed that <it>T. reesei </it>uses primarily the respirative pathway also when growing on the repressive carbon source glucose in contrast to <it>Saccharomyces cerevisiae</it>, which substantially diminishes the respirative pathway flux under glucose repression.</p

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4–18GlcNAc2, mostly containing neutral core-type N-glycans with 8–10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4–7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.We thank Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, for financial support through the project AshByofactory (PTDC/EBB-EBI/101985/2008-FCOMP-01-0124-FEDER-009701) and MIT-Portugal Program (Ph.D. grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar). We also thank Dr. Olli Aitio (University of Helsinki) for helpful assistance in the interpretation of the NMR data

Reaction pathways during oxidation of cereal β-glucans

Oxidation of cereal beta-glucans may affect their stability in food products. Generally, polysaccharides oxidise via different pathways leading to chain cleavage or formation of oxidised groups within the polymer chain. In this study, oxidation pathways of oat and barley beta-glucans were assessed with different concentrations of hydrogen peroxide (H2O2) or ascorbic acid (Asc) with ferrous iron (Fe2+) as a catalyst. Degradation of beta-glucans was evaluated using high performance size exclusion chromatography and formation of carbonyl groups using carbazole-9-carbonyloxyamine labelling. Furthermore, oxidative degradation of glucosyl residues was studied. Based on the results, the oxidation with Asc mainly resulted in glycosidic bond cleavage. With H2O2, both glycosidic bond cleavage and formation of carbonyl groups within the beta-glucan chain was found. Moreover, H2O2 oxidation led to production of formic acid, which was proposed to result from Ruff degradation where oxidised glucose (gluconic acid) is decarboxylated to form arabinose. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in “hairy root” organ cultures of R. strica

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (H-1 NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of H-1 NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in "hairy root" organ cultures of R. strica.Peer reviewe

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in “hairy root” organ cultures of R. strica

A multi-level study of recombinant Pichia pastoris in different oxygen conditions

Background: Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism. Results: The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking. Conclusions: This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion