The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana
Gonzalez-Cabanelas, D. ; Wright, L.P. ; Paetz, C. ; Onkokesung, N. ; Gershenzon, J. ; Rodriguez-Concepcion, M. ; Phillips, M.A. - \ 2015
The Plant Journal 82 (2015)1. - ISSN 0960-7412 - p. 122 - 137.
plant defensin gene - isoprenoid biosynthesis - salicylic-acid - gas-chromatography - mass-spectrometry - mep pathway - synthase - resistance - jasmonate - reveals
2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-D-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.
Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae
Jongedijk, E.J. ; Cankar, K. ; Ranzijn, J. ; Krol, A.R. van der; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2015
Yeast 32 (2015)1. - ISSN 0749-503X - p. 159 - 171.
monoterpene biosynthesis - escherichia-coli - synthase - precursor
Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028¿mg/l (+)-limonene and 0.060¿mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12¿mg/l (+)-limonene and 0.49¿mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., 1996); AF514287.1 (Citrus limonene synthase; Lucker et al., 2002).
Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production
Masakapalli, S.K. ; Ritala, A. ; Dong, L.M. ; Krol, A.R. van der; Oksman-Caldentey, K.M. ; Ratcliffe, R.G. ; Sweetlove, L.J. - \ 2014
Phytochemistry 99 (2014). - ISSN 0031-9422 - p. 73 - 85.
heterotrophic arabidopsis cells - central carbon metabolism - alkaloid biosynthesis - mevalonate pathway - mass-spectrometry - synthase - networks - plants - quantification - expression
The goal of this study was to characterise the metabolic flux phenotype of transgenic tobacco (Nicotiana tabacum) hairy roots engineered for increased biosynthesis of geraniol, an intermediate of the terpenoid indole alkaloid pathway. Steady state, stable isotope labelling was used to determine flux maps of central carbon metabolism for transgenic lines over-expressing (i) plastid-targeted geraniol synthase (pGES) from Valeriana officinalis, and (ii) pGES in combination with plastid-targeted geranyl pyrophosphate synthase from Arabidopsis thaliana (pGES + pGPPS), as well as for wild type and control-vector-transformed roots. Fluxes were constrained by the redistribution of label from [1-C-13]-, [2-C-13]- or [C-13(6)]glucose into amino acids, sugars and organic acids at isotopic steady state, and by biomass output fluxes determined from the fractionation of [U-C-14]glucose into insoluble polymers. No significant differences in growth and biomass composition were observed between the lines. The pGES line accumulated significant amounts of geraniol/geraniol glycosides (151 +/- 24 ng/mg dry weight) and the de nova synthesis of geraniol in pGES was confirmed by C-13 labelling analysis. The pGES + pGPPS also accumulated geraniol and geraniol glycosides, but to lower levels than the pGES line. Although there was a distinct impact of the transgenes at the level of geraniol synthesis, other network fluxes were unaffected, reflecting the capacity of central metabolism to meet the relatively modest.demand for increased precursors in the transgenic lines. It is concluded that re-engineering of the terpenoid indole alkaloid pathway will only require simultaneous manipulation of the steps producing the pathway precursors that originate in central metabolism in tissues engineered to produce at least an order of magnitude more geraniol than has been achieved so far. (C) 2013 Elsevier Ltd. All rights reserved.
Assessment of pleiotropic transcriptome perturbations in Arabidopsis engineered for indirect insect defence
Houshyani Hassanzadeh, B. ; Krol, A.R. van der; Bino, R.J. ; Bouwmeester, H.J. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229
global gene-expression - metabolomics - thaliana - gm - biosynthesis - metabolism - emission - synthase - crops - wheat
Background: Molecular characterization is an essential step of risk/safety assessment of genetically modified (GM) crops. Holistic approaches for molecular characterization using omics platforms can be used to confirm the intended impact of the genetic engineering, but can also reveal the unintended changes at the omics level as a first assessment of potential risks. The potential of omics platforms for risk assessment of GM crops has rarely been used for this purpose because of the lack of a consensus reference and statistical methods to judge the significance or importance of the pleiotropic changes in GM plants. Here we propose a meta data analysis approach to the analysis of GM plants, by measuring the transcriptome distance to untransformed wild-types. Results: In the statistical analysis of the transcriptome distance between GM and wild-type plants, values are compared with naturally occurring transcriptome distances in non-GM counterparts obtained from a database. Using this approach we show that the pleiotropic effect of genes involved in indirect insect defence traits is substantially equivalent to the variation in gene expression occurring naturally in Arabidopsis. Conclusion: Transcriptome distance is a useful screening method to obtain insight in the pleiotropic effects of genetic modification.
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
Ritala, A. ; Dong, L. ; Imseng, N. ; Seppanen-Laakso, T. ; Vasilev, N. ; Krol, A.R. van der; Rischer, H. ; Maaheimo, H. ; Virkki, A. ; Brandli, J. ; Schillberg, S. ; Eibl, R. ; Bouwmeester, H.J. ; Oksman-Caldentey, K.M. - \ 2014
Journal of Biotechnology 176 (2014). - ISSN 0168-1656 - p. 20 - 28.
catharanthus-roseus - isoprenoid biosynthesis - plastidial pathways - essential oils - key enzyme - monoterpene - cultures - synthase - cells - bioreactors
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 plastidtargeted 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 g/g dry weight (DW) and a maximum of 31.3 g/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 g/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.
The seco-iridoid pathway from Catharanthus roseus
Miettinen, K. ; Dong, L. ; Navrot, N. ; Burlat, V. ; Schneider, T. ; Pollier, J. ; Woittiez, L.S. ; Krol, A.R. van der; Lugan, R. ; Llc, T. ; Verpoorte, R. ; Oksman-Caldentey, K.M. ; Martinoia, E. ; Bouwmeester, H.J. - \ 2014
Nature Communications 5 (2014). - ISSN 2041-1723 - 12 p.
indole alkaloid biosynthesis - subcellular organization - rauwolfia-serpentina - statistical-model - plants - expression - proteins - synthase - enzyme - cytochrome-p450
The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.
Expression of an amylosucrase gene in potato results in larger starch granules with novel properties
Huang, X. ; Nazarian, F. ; Vincken, J.P. ; Ji, Q. ; Visser, R.G.F. ; Trindade, L.M. - \ 2014
Planta 240 (2014)2. - ISSN 0032-0935 - p. 409 - 421.
antisense inhibition - branching enzyme - tuber - synthase - glycogen - amylopectin - reduction - amylose - sucrose - size
Main conclusion - Expression of amylosucrase in potato resulted in larger starch granules with rough surfaces and novel physico-chemical properties, including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. Starch is a very important carbohydrate in many food and non-food applications. In planta modification of starch by genetic engineering has significant economic and environmental benefits as it makes the chemical or physical post-harvest modification obsolete. An amylosucrase from Neisseria polysaccharea fused to a starch-binding domain (SBD) was introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. Expression of SBD–amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a twofold increase in median granule size, and altered physico-chemical properties including improved freeze–thaw stability, higher end viscosity, and better enzymatic digestibility. These effects are possibly a result of the physical interaction between amylosucrase and starch granules. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze–thaw stability.
Cytochrome P450s from Cynara cardunculus L. CYP71AV9 andCYP71BL5, catalyze distinct hydroxylations in the sesquiterpenelactone biosynthetic pathway
Eljounaidi, K. ; Cankar, K. ; Comino, C. ; Moglia, A. ; Hehn, A. ; Bourgaud, F. ; Bouwmeester, H.J. ; Menin, B. ; Lanteri, S. ; Beekwilder, M.J. - \ 2014
Plant Science 223 (2014). - ISSN 0168-9452 - p. 59 - 68.
artichoke leaf extract - globe artichoke - scolymus l. - biological-activities - yeast expression - cynaropicrin - synthase - chicory - (+)-germacrene - costunolide
Cynara cardunculus (Asteraceae) is a cross pollinated perennial crop which includes the two cultivatedtaxa globe artichoke and cultivated cardoon. The leaves of these plants contain high concentrationsof sesquiterpene lactones (STLs) among which cynaropicrin is the most represented, and has recentlyattracted attention because of its therapeutic potential as anti-tumor and anti-photoaging agent. Costuno-lide is considered the common precursor of the STLs and three enzymes are involved in its biosyntheticpathway: i.e. the germacrene A synthase (GAS), the germacrene A oxidase (GAO) and the costunolidesynthase (COS). Here we report on the isolation of two P450 genes, (i.e. CYP71AV9 and CYP71BL5), ina set of ~19,000 C. cardunculus unigenes, and their functional characterization in yeast and in planta.The metabolite analyses revealed that the co-expression of CYP71AV9 together with GAS resulted in thebiosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid in yeast. The co-expression of CYP71BL5 andCYP71AV9 with GAS led to biosynthesis of the free costunolide in yeast and costunolide conjugates inNicotiana benthamiana, demonstrating their involvement in STL biosynthesis as GAO and COS enzymes.The substrate specificity of CYP71AV9 was investigated by testing its ability to convert amorpha-4,11-diene, (+)-germacrene D and cascarilladiene to their oxidized products when co-expressed in yeast withthe corresponding terpene synthases.
Expression of an engineered granule-bound Escherichia coli glycogen branching enzyme in potato results in severe morphological changes in starch granules
Huang, X. ; Nazarian Firouzabadi, F. ; Vincken, J.P. ; Ji, Q. ; Suurs, L.C.J.M. ; Visser, R.G.F. ; Trindade, L.M. - \ 2013
Plant Biotechnology Journal 11 (2013)4. - ISSN 1467-7644 - p. 470 - 479.
binding domain - gene-expression - beta-amylase - freeze-thaw - amylose - biosynthesis - amylopectin - arabidopsis - synthase - protein
The Escherichia coli glycogen branching enzyme (GLGB) was fused to either the C- or N-terminus of a starch-binding domain (SBD) and expressed in two potato genetic backgrounds: the amylose-free mutant (amf) and an amylose-containing line (Kardal). Regardless of background or construct used, a large amount of GLGB/SBD fusion protein was accumulated inside the starch granules, however, without an increase in branching. The presence of GLGB/SBD fusion proteins resulted in altered morphology of the starch granules in both genetic backgrounds. In the amf genetic background, the starch granules showed both amalgamated granules and porous starch granules, whereas in Kardal background, the starch granules showed an irregular rough surface. The altered starch granules in both amf and Kardal backgrounds were visible from the initial stage of potato tuber development. High-throughput transcriptomic analysis showed that expression of GLGB/SBD fusion protein in potato tubers did not affect the expression level of most genes directly involved in the starch biosynthesis except for the up-regulation of a beta-amylase gene in Kardal background. The beta-amylase protein could be responsible for the degradation of the extra branches potentially introduced by GLGB.
Genomic regions associated with bovine milk fatty acids in both summer and winter milk samples
Bouwman, A.C. ; Visker, M.H.P.W. ; Arendonk, J.A.M. van; Bovenhuis, H. - \ 2012
BMC Genetics 13 (2012). - ISSN 1471-2156 - 13 p.
wide association - genetic-parameters - dairy-cattle - bos-taurus - dgat1 - polymorphisms - replication - validation - synthase - cows
Background - In this study we perform a genome-wide association study (GWAS) for bovine milk fatty acids from summer milk samples. This study replicates a previous study where we performed a GWAS for bovine milk fatty acids based on winter milk samples from the same population. Fatty acids from summer and winter milk are genetically similar traits and we therefore compare the regions detected in summer milk to the regions previously detected in winter milk GWAS to discover regions that explain genetic variation in both summer and winter milk. Results - The GWAS of summer milk samples resulted in 51 regions associated with one or more milk fatty acids. Results are in agreement with most associations that were previously detected in a GWAS of fatty acids from winter milk samples, including eight ‘new’ regions that were not considered in the individual studies. The high correlation between the –log10(P-values) and effects of SNPs that were found significant in both GWAS imply that the effects of the SNPs were similar on winter and summer milk fatty acids. Conclusions - The GWAS of fatty acids based on summer milk samples was in agreement with most of the associations detected in the GWAS of fatty acids based on winter milk samples. Associations that were in agreement between both GWAS are more likely to be involved in fatty acid synthesis compared to regions detected in only one GWAS and are therefore worthwhile to pursue in fine-mapping studies.
Short communication: A new bovine milk-protein variant: a-lactalbumin variant D.
Visker, M.H.P.W. ; Heck, J.M. ; Valenberg, H.J.F. van; Arendonk, J.A.M. van; Bovenhuis, H. - \ 2012
Journal of Dairy Science 95 (2012)4. - ISSN 0022-0302 - p. 2165 - 2169.
single-base polymorphisms - lactose synthetase - production traits - region - gene - cows - sequence - synthase - cattle
Capillary zone electrophoresis of 1,948 Holstein-Friesian cows suggested the presence of an unknown protein variant of Î±-lactalbumin (Î±-LA) in the milk of 1 cow. Sequencing genomic DNA of this cow showed a polymorphism in the Î±-LA gene (LAA) that appears to be responsible for this protein variant. This single nucleotide polymorphism g.600G > T was located in exon 2 of LAA and causes the amino acid change 65Gln > His in the Î±-LA protein. This Î±-LA protein variant is a new protein variant and should be called Î±-LA protein variant D. This amino acid change is not expected to affect protein function. Genomic DNA of 156 bulls of various dairy cattle breeds was screened to examine the presence of the new Î±-LA protein variant D. Single nucleotide polymorphism g.600G > T, responsible for Î±-LA protein variant D, was not found in any of the 156 bulls. However, 10 other polymorphisms in the coding and promoter regions of LAA were detected that were used to construct haplotypes.
A limited set of starch related genes explain several interrelated traits in potato
Werij, J.S. ; Furrer-Verhorst, M. ; Eck, H.J. van; Visser, R.G.F. ; Bachem, C.W.B. - \ 2012
Euphytica 186 (2012)2. - ISSN 0014-2336 - p. 501 - 516.
adp-glucose pyrophosphorylase - physicochemical properties - diploid potato - quality traits - water dikinase - alpha-glucan - amylose-free - tuber - inhibition - synthase
To understand the molecular basis of potato starch related traits and the underlying starch biosynthesis and degradation, a Quantitative Trait Locus (QTL) analysis in combination with a candidate gene approach was performed. The diploid mapping population C × E, consisting of 249 individuals, was assayed over two consecutive years, for chipping colour, cold induced sweetening, starch content, starch granule size, starch gelling temperature, starch enthalpy, amylose content and degree of starch phosphorylation. QTLs were observed for all traits, except enthalpy on eight out of the twelve potato chromosomes. Several QTLs were found to be consistent over 2 years. Clustering of co-localizing QTLs was observed on some chromosomes, indicating common genetic factors for the different traits. On chromosome 2, Soluble Starch Synthase 2 mapped on the same position as QTLs for starch phosphorylation, starch gelling temperature and amylose content. a-glucan, water dikinase co-localizes on chromosome 5 together with QTLs for starch phosphorylation and cold induced sweetening. Furthermore, the genes coding for two phosphorylases (StPho1a and StPho2) coincide with QTLs for starch gelling temperature, chipping colour and starch granule size on chromosome 2 and a QTL for starch phosphorylation on chromosome 9, respectively. The results suggest allelic variation acting on the genetics of the different traits
Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases
Chavaroche, A.A.E. ; Broek, L.A.M. van den; Boeriu, C.G. ; Eggink, G. - \ 2012
Applied Microbiology and Biotechnology 95 (2012)5. - ISSN 0175-7598 - p. 1199 - 1210.
heparan-sulfate polymerization - molecular-weight heparin - chemoenzymatic synthesis - hyaluronic-acid - capsular polysaccharide - glycosaminoglycans - identification - synthase - glycosyltransferase - biosynthesis
Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA+) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc+) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA+ and PmHS2-GlcNAc+ is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.
Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia
Koehorst-van Putten, H.J.J. ; Sudarmonowati, E. ; Herman, M. ; Pereira-Bertram, I.J. ; Wolters, A.M.A. ; Meima, H. ; Vetten, N. de; Raemakers, C.J.J.M. ; Visser, R.G.F. - \ 2012
Transgenic Research 21 (2012)1. - ISSN 0962-8819 - p. 39 - 50.
manihot-esculenta crantz - cyclic somatic embryogenesis - t-dna - potato - transformation - plants - regeneration - expression - synthase - cultures
The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.
A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene
Cankar, K. ; Houwelingen, A.M.M.L. van; Bosch, H.J. ; Sonke, Th. ; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2011
FEBS Letters 585 (2011)1. - ISSN 0014-5793 - p. 178 - 182.
functional-characterization - yeast expression - nootkatone - valencene - biosynthesis - sesquiterpenes - artemisinin - synthase - cloning
Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene
Sugar-Mediated Acclimation: The Importance of Sucrose Metabolism in Meristems
Carpentier, S.C. ; Vertommen, A. ; Swennen, R. ; Witters, E. ; Fortes, C. ; Souza, M.T. ; Panis, B. - \ 2010
Journal of Proteome Research 9 (2010)10. - ISSN 1535-3893 - p. 5038 - 5046.
cell-suspension cultures - growing potato-tubers - low internal oxygen - chenopodium-rubrum - gene-expression - osmotic-stress - banana musa - synthase - plants - cryopreservation
We have designed an in vitro experimental setup to study the role of sucrose in sugar-mediated acclimation of banana meristems using established highly proliferating meristem cultures. It is a first step toward the systems biology of a meristem and the understanding of how it can survive severe abiotic stress. Using the 2D-DIGE proteomic approach and a meristem-specific EST library, we describe the long-term acclimation response of banana meristems (after 2, 4, 8, and 14 days) and analyze the role of sucrose in this acclimation by setting up a control, a sorbitol, and a sucrose acclimation treatment over time. Sucrose synthase is the dominant enzyme for sucrose breakdown in meristem tissue, which is most likely related to its lower energy consumption. Metabolizing sucrose is of paramount importance to survive, but the uptake of sugar and its metabolism also drive respiration, which may result in limited oxygen levels. According to our data, a successful acclimation is correlated to an initial efficient uptake of sucrose and subsequently a reduced breakdown of sucrose and an induction of fermentation likely by a lack of oxygen.
Some Phenolic Compounds Increase the Nitric Oxide Level in Endothelial Cells in Vitro
Appeldoorn, M.M. ; Venema, D.P. ; Peters, T.H.F. ; Koenen, M.E. ; Arts, I.C.W. ; Vincken, J.P. ; Gruppen, H. ; Keijer, J. ; Hollman, P.C.H. - \ 2009
Journal of Agricultural and Food Chemistry 57 (2009)17. - ISSN 0021-8561 - p. 7693 - 7699.
rat thoracic aorta - dependent relaxation - red wine - blood-pressure - peanut skins - grape seeds - green tea - flavonoids - synthase - cocoa
The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using the hybrid human endothelial cell line EA.hy926 to rapidly screen phenolic compounds for their NO-stimulating potential. The assay was optimized, and a selection of 33 phenolics, namely, procyanidins, monomeric flavan-3-ols, flavonols, a flavone, a flavanone, a chalcone, a stilbene, and phenolic acids, was tested for their ability to enhance endothelial NO level. Resveratrol, a well-known enhancer of NO level, was included as a positive control. Of the 33 phenolics tested, only resveratrol (285% increase in NO level), quercetin (110% increase), epicatechingallate (ECg) (85% increase), and epigallocatechingallate (EGCg) (60% increase) were significant (P = 0.05) enhancers. Procyanidins showed a nonsignificant tendency to elevate NO level. Concentration-dependent correlations between enhanced NO level and endothelial nitric oxide synthase (eNOS) expression were demonstrated for the three polyphenols tested (resveratrol, ECg, and EGCg). Thus, an easy screening tool for change in cellular NO level was developed. Use of this assay showed that only a limited number of phenolic compounds might enhance NO level with an increased amount of eNOS enzyme as a possible contributing mechanism.
Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis
Kooy, F.K. ; Muyuan Ma, ; Beeftink, H.H. ; Eggink, G. ; Tramper, J. ; Boeriu, C.G. - \ 2009
Analytical Biochemistry 384 (2009)2. - ISSN 0003-2697 - p. 329 - 336.
polyacrylamide-gel electrophoresis - synthase - oligosaccharides - microanalysis - acid - fragments - sulfate - assay - face
Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis
Cellulose microfibril deposition: coordinated activity at the plant plasma membrane
Lindeboom, J.J. ; Mulder, B. ; Vos, J.W. ; Ketelaar, M.J. ; Emons, A.M.C. - \ 2008
Journal of Microscopy 231 (2008)2. - ISSN 0022-2720 - p. 192 - 200.
cortical microtubule arrays - cell-wall - root hairs - equisetum-hyemale - arabidopsis root - calcofluor white - cytochalasin-d - pollen tubes - in-vitro - synthase
Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules
Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria x ananassa).
Almeida, J.R. ; Amico, E. d'; Preuss, A. ; Carbone, F. ; Vos, C.H. de - \ 2007
Archives of Biochemistry and Biophysics 465 (2007)1. - ISSN 0003-9861 - p. 61 - 71.
dihydroflavonol 4-reductases - leucoanthocyanidin reductase - anthocyanidin reductases - heterologous expression - antioxidant activity - molecular-cloning - quality traits - synthase - identification - leaves
The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria x ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products. (c) 2007 Elsevier Inc. All rights reserved.