112 results on '"Maaheimo H"'
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2. S17.9 Enzyme-aided synthesis and NMR-studies ofO-linked type oligosaccharides in reducing form
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Maaheimo, H., Pentflä, L., and Renkonen, O.
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- 1993
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3. Expression of two early genes of the terpenoid biosynthetic pathway in tobacco results in major suppression of the pathway and profound changes in non-target metabolites in a differentiation state dependent manner
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Miralpeix B, Choi YH, Ritala A, Maaheimo H, Capell T, Oksman-Caldentey KM, Verpoorte R, and Christou P
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- 2013
4. Investigating the potential interactions between energy metabolism and recombinant protein production in Pichia pastoris by 13C-based metabolic flux analysis
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Jordà, J., primary, Jouhten, P., additional, Maaheimo, H., additional, Ferrer, P., additional, and Albiol, J., additional
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- 2009
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5. Human 3-fucosyltransferases convert chitin oligosaccharides to products containing a GlcNAc 1-4(Fuc 1-3)GlcNAc 1-4R determinant at the nonreducing terminus
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Natunen, J., primary, Aitio, O., additional, Helin, J., additional, Maaheimo, H., additional, Niemela, R., additional, Heikkinen, S., additional, and Renkonen, O., additional
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- 2001
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6. The β1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone
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Helin, J, primary, Penttilä, L, additional, Leppänen, A, additional, Maaheimo, H, additional, Lauri, S, additional, Costello, C.E, additional, and Renkonen, O, additional
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- 1997
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7. Human alpha3-fucosyltransferases convert chitin oligosaccharides to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus.
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Natunen, J, Aitio, O, Helin, J, Maaheimo, H, Niemelä, R, Heikkinen, S, and Renkonen, O
- Abstract
Human alpha3-fucosyltransferases (Fuc-Ts) are known to convert N-acetyllactosamine to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis x antigen); some of them transfer fucose also to GalNAcbeta1-4GlcNAc, generating GalNAcbeta1-4(Fucalpha1-3)GlcNAc determinants. Here, we report that recombinant forms of Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk converted chitin oligosaccharides of 2-4 GlcNAc units efficiently to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus. The product structures were identified by mass spectrometry and nuclear magnetic resonance experiments; rotating frame nuclear Overhauser spectroscopy data suggested that the fucose and the distal N-acetylglucosamine are stacked in the same way as the fucose and the distal galactose of the Lewis x determinant. The products closely resembled a nodulation factor of Mesorhizobium loti but were distinct from nodulation signals generated by NodZ-enzyme.
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- 2001
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8. Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines.
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Niemelä, R, Natunen, J, Penttilä, L, Salminen, H, Helin, J, Maaheimo, H, Costello, C E, and Renkonen, O
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We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain beta1,6-GlcNAc transferase (Leppänen et al., Biochemistry, 36, 13729-13735, 1997).
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- 1999
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9. Enzymatic synthesis of site-specifically (a 1-3)-fucosylated polylactosamines containing either a sialyl Lewis x, a VIM-2, or a sialylated and internally difucosylated sequence
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Raebinae, J., Natunen, J., Niemelae, R., Salminen, H., Ilves, K., Aitio, O., Maaheimo, H., Helin, J., and Renkonen, O.
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- 1997
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10. ^1H and ^1^3C NMR analysis of the pentasaccharide Gal (1 -> 4)GlcNAc (1 -> 3)-[GlcNAc (1 -> 6)]Gal (1 -> 4)GlcNAc synthesized by the mid-chain -(1 -> 6)-D-N-acetylglucosaminyltransferase of rat serum
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Maaheimo, H., Raebinae, J., and Renkonen, O.
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- 1997
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11. Complementary acceptor and site specificities of Fuc-TIV and Fuc-TVII allow effective biosynthesis of sialyl-TriLex and related polylactosamines present on glycoprotein counterreceptors of selectins.
- Author
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Niemelä, R, Natunen, J, Majuri, M L, Maaheimo, H, Helin, J, Lowe, J B, Renkonen, O, and Renkonen, R
- Abstract
The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha2,3-sialylated, multiply alpha1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte alpha1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of alpha2,3-sialylated acceptors but is ineffective in transfer to the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SAalpha2-3'LNbeta1-3'LNbeta1-3'LN (where SA is sialic acid) into the trifucosylated molecule SAalpha2-3'Lexbeta1-3'Lexbeta1-3'Lex (where Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.
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- 1998
12. The 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone
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Helin, J., Penttilae, L., Leppaenen, A., Maaheimo, H., Lauri, S., Costello, C. E., and Renkonen, O.
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- 1997
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13. Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones.
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Leppänen, A, Zhu, Y, Maaheimo, H, Helin, J, Lehtonen, E, and Renkonen, O
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Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.
- Published
- 1998
14. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures
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Jordà Joel, Jouhten Paula, Cámara Elena, Maaheimo Hannu, Albiol Joan, and Ferrer Pau
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Microbiology ,QR1-502 - Abstract
Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.
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- 2012
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15. An analytic and systematic framework for estimating metabolic flux ratios from 13C tracer experiments
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Zamboni Nicola, Jouhten Paula, Rousu Juho, Rantanen Ari, Maaheimo Hannu, and Ukkonen Esko
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Computer applications to medicine. Medical informatics ,R858-859.7 ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Metabolic fluxes provide invaluable insight on the integrated response of a cell to environmental stimuli or genetic modifications. Current computational methods for estimating the metabolic fluxes from 13C isotopomer measurement data rely either on manual derivation of analytic equations constraining the fluxes or on the numerical solution of a highly nonlinear system of isotopomer balance equations. In the first approach, analytic equations have to be tediously derived for each organism, substrate or labelling pattern, while in the second approach, the global nature of an optimum solution is difficult to prove and comprehensive measurements of external fluxes to augment the 13C isotopomer data are typically needed. Results We present a novel analytic framework for estimating metabolic flux ratios in the cell from 13C isotopomer measurement data. In the presented framework, equation systems constraining the fluxes are derived automatically from the model of the metabolism of an organism. The framework is designed to be applicable with all metabolic network topologies, 13C isotopomer measurement techniques, substrates and substrate labelling patterns. By analyzing nuclear magnetic resonance (NMR) and mass spectrometry (MS) measurement data obtained from the experiments on glucose with the model micro-organisms Bacillus subtilis and Saccharomyces cerevisiae we show that our framework is able to automatically produce the flux ratios discovered so far by the domain experts with tedious manual analysis. Furthermore, we show by in silico calculability analysis that our framework can rapidly produce flux ratio equations – as well as predict when the flux ratios are unobtainable by linear means – also for substrates not related to glucose. Conclusion The core of 13C metabolic flux analysis framework introduced in this article constitutes of flow and independence analysis of metabolic fragments and techniques for manipulating isotopomer measurements with vector space techniques. These methods facilitate efficient, analytic computation of the ratios between the fluxes of pathways that converge to a common junction metabolite. The framework can been seen as a generalization and formalization of existing tradition for computing metabolic flux ratios where equations constraining flux ratios are manually derived, usually without explicitly showing the formal proofs of the validity of the equations.
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- 2008
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16. Stepwise transfer of a-D-Galp-(1 -> 3)- -D-Galp-(1 -> 4)- -D-GlcpNAc sequences to 3-OH and 6-OH of distal galactose residues in bi-, tri-, and tetra-antennary asialo-glycans of N-linked complex type
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Helin, J., Maaheimo, H., Seppo, A., and Keane, A.
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- 1995
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17. Enzyme-aided construction of medium-sized alditols of complete O-linked saccharides: The constructed hexasaccharide alditol Gal 1-4GlcNAc 1-6Gal 1-4GlcNac 1-6(Gal 1-3)GalNAc-ol resists the action of endo- -galactosidase from Bacteroides fragilis
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Maaheimo, H., Penttilae, L., and Renkonen, O.
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- 1994
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18. S17.1 Enzyme-aidedde novosynthesis of an octadecamericN-acetylactosaminoglycan
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Renkonen, O., Pentfld, L., Niemelä, R., Seppo, A., Maaheimo, H., Helin, J., Leppdnen, A., and Vilkman, A.
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- 1993
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19. Conformational analysis of three oligosaccharides related to the branch region of multivalent sLex Glycans
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Maaheimo, H, Aitio, O, Taskinen, J, and Renkonen, O
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- 1997
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20. Investigating the potential interactions between energy metabolism and recombinant protein production in Pichia pastoris by 13C-based metabolic flux analysis
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Jordà, J., Jouhten, P., Maaheimo, H., Ferrer, P., and Albiol, J.
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- 2009
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21. Glycoside Phosphorylase Catalyzed Cellulose and β-1,3-Glucan Synthesis Using Chromophoric Glycosyl Acceptors.
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Pylkkänen R, Maaheimo H, Liljeström V, Mohammadi P, and Penttilä M
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- Clostridium thermocellum enzymology, Phosphorylases metabolism, Phosphorylases chemistry, Cellulose chemistry, beta-Glucans chemistry, beta-Glucans metabolism, Glucosyltransferases chemistry, Glucosyltransferases metabolism
- Abstract
Glycoside phosphorylases are enzymes that are frequently used for polysaccharide synthesis. Some of these enzymes have broad substrate specificity, enabling the synthesis of reducing-end-functionalized glucan chains. Here, we explore the potential of glycoside phosphorylases in synthesizing chromophore-conjugated polysaccharides using commercially available chromophoric model compounds as glycosyl acceptors. Specifically, we report cellulose and β-1,3-glucan synthesis using 2-nitrophenyl β-d-glucopyranoside, 4-nitrophenyl β-d-glucopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl β-d-glucopyranoside with Clostridium thermocellum cellodextrin phosphorylase and Thermosipho africanus β-1,3-glucan phosphorylase as catalysts. We demonstrate activity for both enzymes with all assayed chromophoric acceptors and report the crystallization-driven precipitation and detailed structural characterization of the synthesized polysaccharides, i.e., their molar mass distributions and various structural parameters, such as morphology, fibril diameter, lamellar thickness, and crystal form. Our results provide insights for the studies of chromophore-conjugated low molecular weight polysaccharides, glycoside phosphorylases, and the hierarchical assembly of crystalline cellulose and β-1,3-glucan.
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- 2024
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22. Halogenation at the Phenylalanine Residue of Monomethyl Auristatin F Leads to a Favorable cis / trans Equilibrium and Retained Cytotoxicity.
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Sokka IK, Imlimthan S, Sarparanta M, Maaheimo H, Johansson MP, and Ekholm FS
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- Aminobenzoates chemistry, Animals, Cell Line, Tumor, Cell Survival drug effects, Humans, Isomerism, Magnetic Resonance Spectroscopy methods, Mice, Molecular Conformation, Neoplasms pathology, Antineoplastic Agents chemistry, Cytotoxins chemistry, Halogenation, Immunoconjugates chemistry, Neoplasms metabolism, Oligopeptides chemistry, Phenylalanine chemistry
- Abstract
Halogenation can be utilized for the purposes of labeling and molecular imaging, providing a means to, e.g., follow drug distribution in an organism through positron emission tomography (PET) or study the molecular recognition events unfolding by nuclear magnetic resonance (NMR) spectroscopy. For cancer therapeutics, where often highly toxic substances are employed, it is of importance to be able to track the distribution of the drugs and their metabolites in order to ensure minimal side effects. Labeling should ideally have a negligible disruptive effect on the efficacy of a given drug. Using a combination of NMR spectroscopy and cytotoxicity assays, we identify a site susceptible to halogenation in monomethyl auristatin F (MMAF), a widely used cytotoxic agent in the antibody-drug conjugate (ADC) family of cancer drugs, and study the effects of fluorination and chlorination on the physiological solution structure of the auristatins and their cytotoxicity. We find that the cytotoxicity of the parent drug is retained, while the conformational equilibrium is shifted significantly toward the biologically active trans isomer, simultaneously decreasing the concentration of the inactive and potentially disruptive cis isomer by up to 50%. Our results may serve as a base for the future assembly of a multifunctional toolkit for the assessment of linker technologies and exploring bystander effects from the warhead perspective in auristatin-derived ADCs.
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- 2021
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23. Production of D-lactic acid containing polyhydroxyalkanoate polymers in yeast Saccharomyces cerevisiae.
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Ylinen A, Maaheimo H, Anghelescu-Hakala A, Penttilä M, Salusjärvi L, and Toivari M
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- Acetyl-CoA C-Acetyltransferase genetics, Acetyl-CoA C-Acetyltransferase metabolism, Acyltransferases genetics, Acyltransferases metabolism, Alcohol Oxidoreductases genetics, Alcohol Oxidoreductases metabolism, Coenzyme A-Transferases genetics, Coenzyme A-Transferases metabolism, Escherichia coli metabolism, Genetic Engineering, Hydroxybutyrates metabolism, Industrial Microbiology, Metabolic Networks and Pathways, Polyhydroxyalkanoates chemistry, Lactic Acid metabolism, Polyhydroxyalkanoates biosynthesis, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Polyhydroxyalkanoates (PHAs) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6-19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight (CDW). According to NMR, P(LA-3HB) contained D-lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the CDW. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6, 6.3, and 1 130 kDa for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)
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- 2021
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24. Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho -Carboranylmethyl-Bearing Glucoconjugates.
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Matović J, Järvinen J, Sokka IK, Imlimthan S, Raitanen JE, Montaser A, Maaheimo H, Huttunen KM, Peräniemi S, Airaksinen AJ, Sarparanta M, Johansson MP, Rautio J, and Ekholm FS
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- Boron Compounds administration & dosage, Boron Compounds chemistry, Boron Neutron Capture Therapy methods, Cell Line, Tumor, Glucose metabolism, Humans, Boron administration & dosage, Boron chemistry, Brain Neoplasms radiotherapy, Glucose Transporter Type 1 metabolism
- Abstract
Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of ortho -carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.
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- 2021
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25. Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods.
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Voutilainen S, Heinonen M, Andberg M, Jokinen E, Maaheimo H, Pääkkönen J, Hakulinen N, Rouvinen J, Lähdesmäki H, Kaski S, Rousu J, Penttilä M, and Koivula A
- Subjects
- Aldehyde-Lyases genetics, Aldehyde-Lyases metabolism, Machine Learning, Protein Engineering, Substrate Specificity, Escherichia coli genetics, Escherichia coli metabolism, Fructose-Bisphosphate Aldolase genetics
- Abstract
In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. KEY POINTS: • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.
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- 2020
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26. Production and characterization of Aspergillus niger GH29 family α-fucosidase and production of a novel non-reducing 1-fucosyllactose.
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Usvalampi A, Ruvalcaba Medrano M, Maaheimo H, Salminen H, Tossavainen O, and Frey AD
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- Enzyme Stability, Fucose analogs & derivatives, Fucose metabolism, Lactose analogs & derivatives, Lactose metabolism, Substrate Specificity, Aspergillus niger enzymology, Fungal Proteins metabolism, alpha-L-Fucosidase metabolism
- Abstract
Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60 °C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45 °C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the K
M and 2.8 mmol/(mg*h) for the Vmax . Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the β-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2'-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.- Published
- 2020
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27. Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures.
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Akhgari A, Laakso I, Maaheimo H, Choi YH, Seppänen-Laakso T, Oksman-Caldentey KM, and Rischer H
- Abstract
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 (
1 H 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) of1 H 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.- Published
- 2019
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28. Effect of hydrothermal pretreatment severity on lignin inhibition in enzymatic hydrolysis.
- Author
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Kellock M, Maaheimo H, Marjamaa K, Rahikainen J, Zhang H, Holopainen-Mantila U, Ralph J, Tamminen T, Felby C, and Kruus K
- Subjects
- Adsorption, Biomass, Cellulase metabolism, Cellulose chemistry, Cellulose 1,4-beta-Cellobiosidase metabolism, Hydrolysis, Triticum chemistry, Lignin metabolism
- Abstract
Hydrothermal pretreatment is commonly used for enhancing enzymatic hydrolysis of lignocellulosics. Spruce and wheat straw were pretreated with increasing severity and lignin characteristics were analysed. The effect of enzymatically isolated lignin on the hydrolysis of Avicel and the adsorption of a cellobiohydrolase onto lignin was measured. Non-pretreated lignins had only a minor effect on Avicel hydrolysis. The structural changes in lignin accompanying hydrothermal pretreatment were associated with increased binding and inactivation of the cellulase on the lignin surface. The inhibitory effect was more pronounced in spruce than in wheat straw lignin. However, similar pretreatment severities caused similar levels of inhibition in Avicel hydrolysis for both biomass sources. The combined severity factor of the pretreatment correlated well with the inhibitory effect of lignin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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29. In vitro reconstitution and characterisation of the oxidative D-xylose pathway for production of organic acids and alcohols.
- Author
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Boer H, Andberg M, Pylkkänen R, Maaheimo H, and Koivula A
- Abstract
The oxidative D-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from D-xylose or D-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of D-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family D-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family D-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.
- Published
- 2019
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30. Structural and Functional Insights Into Lysostaphin-Substrate Interaction.
- Author
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Tossavainen H, Raulinaitis V, Kauppinen L, Pentikäinen U, Maaheimo H, and Permi P
- Abstract
Lysostaphin from Staphylococcus simulans and its family enzymes rapidly acquire prominence as the next generation agents in treatment of S. aureus infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity toward pentaglycine bridges in S. aureus cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase. Understanding the determinants affecting the efficiency of catalysis and strength and specificity of interactions lies at the heart of all lysostaphin family enzyme applications. To this end, we have used NMR, SAXS and molecular dynamics simulations to characterize lysostaphin structure and dynamics, to address the inter-domain interaction, the enzyme-substrate interaction as well as the catalytic properties of pentaglycine cleavage in solution. Our NMR structure confirms the recent crystal structure, yet, together with the molecular dynamics simulations, emphasizes the dynamic nature of the loops embracing the catalytic site. We found no evidence for inter-domain interaction, but, interestingly, the SAXS data delineate two preferred conformation subpopulations. Catalytic H329 and H360 were observed to bind a second zinc ion, which reduces lysostaphin pentaglycine cleaving activity. Binding of pentaglycine or its lysine derivatives to the targeting domain was found to be of very low affinity. The pentaglycine interaction site was located to the N-terminal groove of the domain. Notably, the targeting domain binds the peptidoglycan stem peptide Ala-d-γ-Glu-Lys-d-Ala-d-Ala with a much higher, micromolar affinity. Binding site mapping reveals two interaction sites of different affinities on the surface of the domain for this peptide.
- Published
- 2018
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31. Enzymatic synthesis of fucose-containing galacto-oligosaccharides using β-galactosidase and identification of novel disaccharide structures.
- Author
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Usvalampi A, Maaheimo H, Tossavainen O, and Frey AD
- Subjects
- Disaccharides chemistry, Glycosylation, Oligosaccharides chemistry, Disaccharides chemical synthesis, Fucose analogs & derivatives, Galactose analogs & derivatives, beta-Galactosidase metabolism
- Abstract
Fucosylated oligosaccharides have an important role in maintaining a healthy immune system and homeostatic gut microflora. This study employed a commercial β-galactosidase in the production of fucose-containing galacto-oligosaccharides (fGOS) from lactose and fucose. The production was optimized using experiment design and optimal conditions for a batch production in 3-liter scale. The reaction product was analyzed and the produced galactose-fucose disaccharides were purified. The structures of these disaccharides were determined using NMR and it was verified that one major product with the structure Galβ1-3Fuc and two minor products with the structures Galβ1-4Fuc and Galβ1-2Fuc were formed. Additionally, the product composition was defined in more detail using several different analytical methods. It was concluded that the final product contained 42% total monosaccharides, 40% disaccharides and 18% of larger oligosaccharides. 290 μmol of fGOS was produced per gram of reaction mixture and 37% of the added fucose was bound to fGOS. The fraction of fGOS from total oligosaccharides was determined as 44%. This fGOS product could be used as a new putative route to deliver fucose to the intestine.
- Published
- 2018
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32. New insight on the structural features of the cytotoxic auristatins MMAE and MMAF revealed by combined NMR spectroscopy and quantum chemical modelling.
- Author
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Johansson MP, Maaheimo H, and Ekholm FS
- Subjects
- Cell Death drug effects, Molecular Conformation, Thermodynamics, Magnetic Resonance Spectroscopy, Models, Chemical, Oligopeptides chemistry, Oligopeptides pharmacology, Quantum Theory
- Abstract
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.
- Published
- 2017
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33. 13 C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids.
- Author
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Zhang M, Yu XW, Xu Y, Jouhten P, Swapna GVT, Glaser RW, Hunt JF, Montelione GT, Maaheimo H, and Szyperski T
- Subjects
- Aerobiosis physiology, Batch Cell Culture Techniques, Butyrates metabolism, Carbon Isotopes, Citric Acid Cycle physiology, Hemiterpenes, Keto Acids metabolism, Magnetic Resonance Spectroscopy, Mitochondria metabolism, Pentose Phosphate Pathway physiology, Pyruvic Acid metabolism, Saccharomyces cerevisiae metabolism, Glucose metabolism, Isoleucine metabolism, Leucine metabolism, Metabolome physiology, Pichia metabolism, Valine metabolism
- Abstract
Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using
13 C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First,13 C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly2 H,13 C,15 N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology., Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2)., (© 2017 Federation of European Biochemical Societies.)- Published
- 2017
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34. Functional comparison of versatile carbohydrate esterases from families CE1, CE6 and CE16 on acetyl-4-O-methylglucuronoxylan and acetyl-galactoglucomannan.
- Author
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Mai-Gisondi G, Maaheimo H, Chong SL, Hinz S, Tenkanen M, and Master E
- Subjects
- Acetylation, Polysaccharides chemistry, Carbohydrates chemistry, Esterases metabolism, Mannans chemistry, Xylans chemistry
- Abstract
Background: The backbone structure of many hemicelluloses is acetylated, which presents a challenge when the objective is to convert corresponding polysaccharides to fermentable sugars or else recover hemicelluloses for biomaterial applications. Carbohydrate esterases (CE) can be harnessed to overcome these challenges., Methods: Enzymes from different CE families, AnAcXE (CE1), OsAcXE (CE6), and MtAcE (CE16) were compared based on action and position preference towards acetyl-4-O-methylglucuronoxylan (MGX) and acetyl-galactoglucomannan (GGM). To determine corresponding positional preferences, the relative rate of acetyl group released by each enzyme was analyzed by real time
1 H NMR., Results: AnAcXE (CE1) showed lowest specific activity towards MGX, where OsAcXE (CE6) and MtAcE were approximately four times more active than AnAcXE (CE1). MtAcE (CE16) was further distinguished by demonstrating 100 times higher activity on GGM compared to AnAcXE (CE1) and OsAcXE (CE6), and five times higher activity on GGM than MGX. Following 24h incubation, all enzymes removed between 78 and 93% of total acetyl content from MGX and GGM, where MtAcE performed best on both substrates., Major Conclusions: Considering action on MGX, all esterases showed preference for doubly substituted xylopyranosyl residues (2,3-O-acetyl-Xylp). Considering action on GGM, OsAcXE (CE6) preferentially targeted 2-O-acetyl-mannopyranosyl residues (2-O-acetyl-Manp) whereas AnAcXE (CE1) demonstrated highest activity towards 3-O-acetyl-Manp positions; regiopreference of MtAcE (CE16) on GGM was less clear., General Significance: The current comparative analysis identifies options to control the position of acetyl group release at initial stages of reaction, and enzyme combinations likely to accelerate deacetylation of major hemicellulose sources., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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35. Reaction pathways during oxidation of cereal β-glucans.
- Author
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Mäkelä N, Sontag-Strohm T, Schiehser S, Potthast A, Maaheimo H, and Maina NH
- Subjects
- Ascorbic Acid, Edible Grain metabolism, Hydrogen Peroxide, Oxidation-Reduction, Avena metabolism, Hordeum metabolism, beta-Glucans metabolism
- Abstract
Oxidation of cereal β-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 β-glucans were assessed with different concentrations of hydrogen peroxide (H
2 O2 ) or ascorbic acid (Asc) with ferrous iron (Fe2+ ) as a catalyst. Degradation of β-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 H2 O2 , both glycosidic bond cleavage and formation of carbonyl groups within the β-glucan chain was found. Moreover, H2 O2 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)- Published
- 2017
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36. Carbon 13-Metabolic Flux Analysis derived constraint-based metabolic modelling of Clostridium acetobutylicum in stressed chemostat conditions.
- Author
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Wallenius J, Maaheimo H, and Eerikäinen T
- Subjects
- 1-Butanol metabolism, Arabinose metabolism, Butanols metabolism, Carbon Isotopes analysis, Carbon Isotopes metabolism, Clostridium acetobutylicum metabolism, Clostridium acetobutylicum physiology, Metabolic Flux Analysis methods, Models, Biological, Stress, Physiological physiology
- Abstract
The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from (13)C Metabolic Flux Analysis ((13)C-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40n-% rhamnose, 34n-% glucose, 13n-% mannose, 10n-% galactose, and 2n-% arabinose. The EPS was studied to have butanol adsorbing property, 70(butanol)mg(EPS)g(-1) at 37°C., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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37. The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.
- Author
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Biz A, Sugai-Guérios MH, Kuivanen J, Maaheimo H, Krieger N, Mitchell DA, and Richard P
- Subjects
- Aspergillus niger genetics, Beta vulgaris, Citrus, Ethanol metabolism, Fermentation, Fructose metabolism, Hydrolysis, Metabolic Engineering methods, Neurospora crassa genetics, Trichoderma genetics, Hexuronic Acids metabolism, Metabolic Networks and Pathways genetics, Pectins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Background: Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp., Results: In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate., Conclusion: This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.
- Published
- 2016
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38. Optimization of Isomaltooligosaccharide Size Distribution by Acceptor Reaction of Weissella confusa Dextransucrase and Characterization of Novel α-(1→2)-Branched Isomaltooligosaccharides.
- Author
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Shi Q, Hou Y, Juvonen M, Tuomainen P, Kajala I, Shukla S, Goyal A, Maaheimo H, Katina K, and Tenkanen M
- Subjects
- Carbohydrate Sequence, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Proton Magnetic Resonance Spectroscopy, Tandem Mass Spectrometry, Glucosyltransferases metabolism, Oligosaccharides chemistry, Weissella enzymology
- Abstract
Long-chain isomaltooligosaccharides (IMOs) are promising prebiotics. IMOs were produced by a Weissella confusa dextransucrase via maltose acceptor reaction. The inputs of substrates (i.e., sucrose and maltose, 0.15-1 M) and dextransucrase (1-10 U/g sucrose) were used to control IMO yield and profile. According to response surface modeling, 1 M sucrose and 0.5 M maltose were optimal for the synthesis of longer IMOs, whereas the dextransucrase dosage showed no significant effect. In addition to the principal linear IMOs, a homologous series of minor IMOs were also produced from maltose. As identified by MS(n) and NMR spectroscopy, the minor trisaccharide contained an α-(1→2)-linked glucosyl residue on the reducing residue of maltose and thus was α-d-glucopyranosyl-(1→2)-[α-d-glucopyranosyl-(1→4)]-d-glucopyranose (centose). The higher members of the series were probably formed by the attachment of a single unit branch to linear IMOs. This is the first report of such α-(1→2)-branched IMOs produced from maltose by a dextransucrase.
- Published
- 2016
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39. Characterization of a unique Caulobacter crescentus aldose-aldose oxidoreductase having dual activities.
- Author
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Andberg M, Maaheimo H, Kumpula EP, Boer H, Toivari M, Penttilä M, and Koivula A
- Subjects
- Biocatalysis, Caulobacter crescentus metabolism, Glucose metabolism, Nuclear Magnetic Resonance, Biomolecular, Oxidoreductases metabolism, Sequence Homology, Amino Acid, Xylose metabolism, Zymomonas enzymology, Zymomonas metabolism, Aldehyde Reductase metabolism, Caulobacter crescentus enzymology, Monosaccharides metabolism
- Abstract
We describe here the characterization of a novel enzyme called aldose-aldose oxidoreductase (Cc AAOR; EC 1.1.99) from Caulobacter crescentus. The Cc AAOR exists in solution as a dimer, belongs to the Gfo/Idh/MocA family and shows homology with the glucose-fructose oxidoreductase from Zymomonas mobilis. However, unlike other known members of this protein family, Cc AAOR is specific for aldose sugars and can be in the same catalytic cycle both oxidise and reduce a panel of monosaccharides at the C1 position, producing in each case the corresponding aldonolactone and alditol, respectively. Cc AAOR contains a tightly-bound nicotinamide cofactor, which is regenerated in this oxidation-reduction cycle. The highest oxidation activity was detected on D-glucose but significant activity was also observed on D-xylose, L-arabinose and D-galactose, revealing that both hexose and pentose sugars are accepted as substrates by Cc AAOR. The configuration at the C2 and C3 positions of the saccharides was shown to be especially important for the substrate binding. Interestingly, besides monosaccharides, Cc AAOR can also oxidise a range of 1,4-linked oligosaccharides having aldose unit at the reducing end, such as lactose, malto- and cello-oligosaccharides as well as xylotetraose. (1)H NMR used to monitor the oxidation and reduction reaction simultaneously, demonstrated that although D-glucose has the highest affinity and is also oxidised most efficiently by Cc AAOR, the reduction of D-glucose is clearly not as efficient. For the overall reaction catalysed by Cc AAOR, the L-arabinose, D-xylose and D-galactose were the most potent substrates.
- Published
- 2016
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40. Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase.
- Author
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Shi Q, Juvonen M, Hou Y, Kajala I, Nyyssölä A, Maina NH, Maaheimo H, Virkki L, and Tenkanen M
- Subjects
- Dextrans chemistry, Models, Molecular, Cellobiose chemistry, Glucosyltransferases chemistry, Magnetic Resonance Spectroscopy methods, Sucrose chemistry, Tandem Mass Spectrometry methods, Trisaccharides chemistry, Weissella chemistry
- Abstract
Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions of Weissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1 → 2)-linked to the acceptor's reducing end. An additional product, isomelezitose (6(Fru)-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from (13)C6(glc) sucrose and lactose were analyzed by ESI-MS(n), and the fragmentation patterns of these compounds were characterized., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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41. The impact of fermentation with exopolysaccharide producing lactic acid bacteria on rheological, chemical and sensory properties of pureed carrots (Daucus carota L.).
- Author
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Juvonen R, Honkapää K, Maina NH, Shi Q, Viljanen K, Maaheimo H, Virkki L, Tenkanen M, and Lantto R
- Subjects
- Daucus carota chemistry, Food Analysis, Humans, Polysaccharides, Bacterial metabolism, Rheology, Sensation, Daucus carota microbiology, Fermentation, Food Handling methods, Food Microbiology methods, Lactobacillales metabolism
- Abstract
Fermentation with lactic acid bacteria (LAB) offers a natural means to modify technological and nutritional properties of foods and food ingredients. This study explored the impact of fermentation with different exopolysaccharide (EPS) producing LAB on rheological, chemical and sensory properties of puréed carrots in water, as a vegetable model, with the focus on texture formation. The screening of 37 LAB strains for starter selection revealed 16 Lactobacillus, Leuconostoc and Weissella strains capable of EPS (dextran, levan, and/or β-glucan) production in the carrot raw material. Fermentations with five out of six selected EPS producers modified perceived texture of the liquid carrot model (p<0.05). The formation of low-branched dextran correlated with perceived thickness, whereas the production of β-glucan correlated with perceived elasticity. Low-branched dextran producing Weissella confusa and Leuconostoc lactis strains produced thick texture accompanied by pleasant odour and flavour. The fermentation with the selected EPS-producing LAB strains is a promising clean label approach to replace hydrocolloid additives as texturizers in vegetable containing products, not only carrot., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
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42. L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.
- Author
-
Aro-Kärkkäinen N, Toivari M, Maaheimo H, Ylilauri M, Pentikäinen OT, Andberg M, Oja M, Penttilä M, Wiebe MG, Ruohonen L, and Koivula A
- Subjects
- Cloning, Molecular, Coenzymes metabolism, Enzyme Stability, Galactose Dehydrogenases chemistry, Galactose Dehydrogenases genetics, Galactose Dehydrogenases isolation & purification, Gene Expression, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, NAD metabolism, NADP metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Rhizobium leguminosarum metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Sequence Analysis, DNA, Arabinose metabolism, Galactose Dehydrogenases metabolism, Rhizobium leguminosarum enzymology, Saccharomyces cerevisiae metabolism, Sugar Acids metabolism
- Abstract
Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast.
- Published
- 2014
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43. Impact of steam explosion on the wheat straw lignin structure studied by solution-state nuclear magnetic resonance and density functional methods.
- Author
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Heikkinen H, Elder T, Maaheimo H, Rovio S, Rahikainen J, Kruus K, and Tamminen T
- Subjects
- Hot Temperature, Magnetic Resonance Spectroscopy, Molecular Structure, Steam, Lignin chemistry, Plant Stems chemistry, Triticum chemistry
- Abstract
Chemical changes of lignin induced by the steam explosion (SE) process were elucidated. Wheat straw was studied as the raw material, and lignins were isolated by the enzymatic mild acidolysis lignin (EMAL) procedure before and after the SE treatment for analyses mainly by two-dimensional (2D) [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] and (31)P nuclear magnetic resonance (NMR). The β-O-4 structures were found to be homolytically cleaved, followed by recoupling to β-5 linkages. The homolytic cleavage/recoupling reactions were also studied by computational methods, which verified their thermodynamic feasibility. The presence of the tricin bound to wheat straw lignin was confirmed, and it was shown to participate in lignin reactions during the SE treatment. The preferred homolytic β-O-4 cleavage reaction was calculated to follow bond dissociation energies: G-O-G (guaiacyl) (69.7 kcal/mol) > G-O-S (syringyl) (68.4 kcal/mol) > G-O-T (tricin) (67.0 kcal/mol).
- Published
- 2014
- Full Text
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44. Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae.
- Author
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Nygård Y, Maaheimo H, Mojzita D, Toivari M, Wiebe M, Resnekov O, Gustavo Pesce C, Ruohonen L, and Penttilä M
- Subjects
- Computer Simulation, Gene Expression Regulation, Enzymologic physiology, Gene Expression Regulation, Fungal physiology, Signal Transduction physiology, Metabolic Flux Analysis methods, Models, Biological, Saccharomyces cerevisiae metabolism, Sugar Acids metabolism, Xylosidases metabolism
- Abstract
D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by (1)H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo(1)H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision. During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
45. O-acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants.
- Author
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Chong SL, Virkki L, Maaheimo H, Juvonen M, Derba-Maceluch M, Koutaniemi S, Roach M, Sundberg B, Tuomainen P, Mellerowicz EJ, and Tenkanen M
- Subjects
- Acetylation, Arabidopsis enzymology, Arabidopsis genetics, Gene Expression Regulation, Plant, Glycosyltransferases genetics, Glycosyltransferases metabolism, Mutation, Xylans chemistry, Xylans metabolism, Glycosyltransferases biosynthesis, Xylans biosynthesis
- Abstract
O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.
- Published
- 2014
- Full Text
- View/download PDF
46. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.
- Author
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Ritala A, Dong L, Imseng N, Seppänen-Laakso T, Vasilev N, van der Krol S, Rischer H, Maaheimo H, Virkki A, Brändli J, Schillberg S, Eibl R, Bouwmeester H, and Oksman-Caldentey KM
- Subjects
- Acyclic Monoterpenes, DNA, Plant, Phosphoric Monoester Hydrolases genetics, Plant Proteins genetics, Plant Roots enzymology, Plant Roots genetics, Plants, Genetically Modified enzymology, Plants, Genetically Modified genetics, Plants, Genetically Modified metabolism, Secologanin Tryptamine Alkaloids metabolism, Secondary Metabolism, Nicotiana enzymology, Nicotiana genetics, Bioreactors, Phosphoric Monoester Hydrolases metabolism, Plant Proteins metabolism, Plant Roots metabolism, Terpenes metabolism, Nicotiana metabolism, Valerian genetics
- Abstract
The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
47. Labelling analysis for ¹³C MFA using NMR spectroscopy.
- Author
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Jouhten P and Maaheimo H
- Subjects
- Amino Acids analysis, Carbon Isotopes metabolism, Isotope Labeling methods, Magnetic Resonance Spectroscopy methods, Metabolic Flux Analysis methods
- Abstract
NMR spectroscopy is an efficient method for analyzing (13)C labelling of cellular metabolites. The strength of it is especially the ability to provide direct quantitative positional information on the (13)C labelling status of carbon atoms in metabolites. NMR spectroscopic methods allow also for detection of contiguously (13)C-labelled fragments in the carbon backbones of the metabolites. Furthermore, the recent developments of NMR spectroscopy hardware have substantially improved the sensitivity of the methods. In this chapter we describe a method for analyzing the (13)C labelling of the biomass amino acids for metabolic flux analysis, sample preparation for NMR spectroscopy, acquiring and processing the NMR spectra, and extracting the (13)C labelling information from the NMR data. Different NMR methods are applied depending on the (13)C labelling strategy chosen. These strategies include uniform (13)C labelling, positional (13)C labelling, or a combination of both. Not only the preparation of sample for analysis of (13)C labelling in proteinogenic amino acids in biomass is described, but also the necessary modifications to the method when analysis of (13)C labelling in free metabolic intermediates is of interest. Finally the strategies for using the different NMR-detected (13)C labelling data in (13)C MFA are discussed.
- Published
- 2014
- Full Text
- View/download PDF
48. Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.
- Author
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Aguiar TQ, Maaheimo H, Heiskanen A, Wiebe MG, Penttilä M, and Domingues L
- Subjects
- Eremothecium metabolism, Glycosylation, Polysaccharides chemistry, Eremothecium chemistry, Polysaccharides biosynthesis, Polysaccharides metabolism
- Abstract
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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
49. Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides.
- Author
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Mikkelson A, Maaheimo H, and Hakala TK
- Subjects
- Chromatography, Gel, Chromatography, High Pressure Liquid, Hydrolysis, Magnetic Resonance Spectroscopy, Mannans chemistry, Mannose chemistry, Oligosaccharides isolation & purification, Cellulase metabolism, Mannans metabolism, Mannosidases metabolism, Oligosaccharides chemical synthesis, Trichoderma enzymology
- Abstract
In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86-95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
50. Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate.
- Author
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Toivari M, Nygård Y, Kumpula EP, Vehkomäki ML, Benčina M, Valkonen M, Maaheimo H, Andberg M, Koivula A, Ruohonen L, Penttilä M, and Wiebe MG
- Subjects
- Alcohol Oxidoreductases biosynthesis, Alcohol Oxidoreductases genetics, Aldehyde Reductase genetics, Aldehyde Reductase metabolism, Animals, Caulobacter crescentus enzymology, Caulobacter crescentus genetics, Ethanol metabolism, Glucose metabolism, Liver enzymology, Saccharomyces cerevisiae genetics, Swine metabolism, Metabolic Engineering methods, Saccharomyces cerevisiae metabolism, Uronic Acids metabolism, Xylose metabolism
- Abstract
An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ∼70 mgg(-1); xylitol to ∼18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1)., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
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