Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Folate and epigenetics : Why we should not forget bacterial biosynthesis
Kok, Dieuwertje E. ; Steegenga, Wilma T. ; McKay, Jill A. - \ 2018
Epigenomics 10 (2018)9. - ISSN 1750-1911 - p. 1147 - 1150.
biosynthesis - DNA methylation - epigenetics - folate - intestinal bacteria - microbiota - one-carbon metabolism
Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

Production of protein‐based polymers in Pichia pastoris
Werten, Marc W.T. - \ 2017
University. Promotor(en): Martien Cohen Stuart; Gerrit Eggink, co-promotor(en): Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789463436069 - 241
proteins - polymers - pichia pastoris - gelatin - proteolysis - biosynthesis - eiwitten - polymeren - gelatine - proteolyse - biosynthese

From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems.

The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale.

We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to address problems such as proteolytic degradation and other undesired posttranslational modifications. The basic physicochemical properties of the polymers produced were studied, which revealed interesting characteristics. Some of these polymers show promise for further development towards biomedical applications such as tissue engineering and controlled drug release.

The ribosome regulates flavodoxin folding
Houwman, Joseline A. - \ 2017
University. Promotor(en): Willem van Berkel, co-promotor(en): Carlo van Mierlo. - Wageningen : Wageningen University - ISBN 9789463431453 - 176
proteins - flavoproteins - cofactors - ribosomes - biosynthesis - eiwitten - flavoproteinen - co-factoren - ribosomen - biosynthese

During and after their translation by the ribosome, folding of polypeptides to biologically active proteins is of vital importance for all living organisms. Gaining knowledge about nascent chain folding is required to enhance our understanding of protein folding in the cell. This in turn allows us to obtain insights into factors responsible for protein misfolding, aggregation, and, potentially, for numerous devastating pathologies.

In Chapter 1 the model protein flavodoxin is introduced. Also theories about protein folding are presented, which led to the concept of the “folding energy landscape”. Flavodoxin folds via a misfolded off-pathway intermediate, which is molten globular and forms extensively during its refolding in vitro.

In Chapter 2 we show that flavodoxin also populates an on-pathway molten globule during its folding. In the F44Y variant of apoflavodoxin, lowering the ionic strength induces the off-pathway molten globule state. By adding the cofactor FMN, we could follow aspects of the folding of this protein, as off-pathway molten globular flavodoxin first has to unfold and subsequently refold before FMN can bind. Thus, presence of the off-pathway molten globule retards FMN binding. We determined the presence of the off-pathway molten globule at decreasing ionic strengths with cofactor binding kinetics and polarized time-resolved tryptophan fluorescence spectroscopy. Comparison of both data sets revealed the presence of another, concurrently present molten globule. This species is most likely on-pathway to native protein. To our knowledge this is the first time that two concurrent molten globules have been discovered that reside on folding routes of decidedly different nature (i.e., on- and off-pathway ones).

While much work has been done on the folding of flavodoxin in vitro, the next step is to elucidate how this protein folds in vivo. In Chapter 3 the first insights into cotranslational flavodoxin folding are presented. By using ribosomal nascent chains (RNCs) we could determine that when flavodoxin is fully exposed outside the ribosome it can bind its cofactor. However, while its five C-terminal amino acids are still sequestered in the ribosomal exit tunnel, the protein is in a non-native state and cannot bind FMN. Thus the last step in production of this flavoprotein in vivo is the binding of cofactor.

Chapter 4 reveals the influence of the ribosome on formation of the off-pathway molten globule of flavodoxin. By using RNCs of the F44Y variant of apoflavodoxin, we proved that the ribosome restrains formation of this molten globule. This discovery was possible by exploiting the findings of Chapter 2 and Chapter 3, namely that cofactor binding kinetics slow down when off-pathway molten globule is present and that a fully exposed, natively folded flavodoxin nascent chain binds FMN. For F44Y RNCs no retardation in FMN binding occurs, whereas cofactor binding slows down in case of isolated, full-length F44Y in the molten globule state. Thus the ribosome restrains formation of molten globules in stalled nascent flavodoxin. This is possibly due to electrostatic repulsion of the nascent chain by the ribosomal surface, as both are negatively charged, leading to entropic stabilization of native protein at physiological ionic strength.

In Chapter 5 we review experiments and simulations concerning the folding of flavodoxins and CheY-like proteins, which share the flavodoxin-like fold. These proteins form intermediates that are off-pathway to native protein and several of these species are molten globules. This susceptibility to frustration is caused by the more rapid formation of an α-helix compared to a β-sheet, particularly when a parallel β-sheet is involved. The experimentally characterized off-pathway intermediates seem to be of α-helical nature. We discuss the probing of the cotranslational folding of flavodoxin as a first step towards a molecular description of how flavodoxin-like proteins fold in vivo.

Finally, Chapter 6 touches upon the implications of our findings and possible applications in biotechnology, health and disease. A finding that has potential application is the role FMN has as a chemical chaperone. This chemical chaperone can already work at the cotranslational level, as binding of FMN stabilizes a nascent chain and thereby protects the nascent chain against degradation.

Plant growth promotion by Pseudomonas fluorescens : mechanisms, genes and regulation
Cheng, X. - \ 2016
University. Promotor(en): Francine Govers; J.M. Raaijmakers, co-promotor(en): M. van der Voort. - Wageningen : Wageningen University - ISBN 9789462578753 - 192 p.
soil bacteria - pseudomonas fluorescens - plants - growth stimulators - soil suppressiveness - plant diseases - induced resistance - biochemistry - biosynthesis - plant-microbe interactions - transcriptomics - bodembacteriën - planten - groeistimulatoren - bodemweerbaarheid - plantenziekten - geïnduceerde resistentie - biochemie - biosynthese - plant-microbe interacties - transcriptomica

Pseudomonas fluorescens is a Gram-negative rod shaped bacterium that has a versatile metabolism and is widely spread in soil and water. P. fluorescens strain SBW25 (Pf.SBW25) is a well-known model strain to study bacterial evolution, plant colonization and biocontrol of plant diseases. It produces the biosurfactant viscosin, a lipopeptide that plays a key role in motility, biofilm formation and activity against zoospores of Phytophthora infestans and other oomycete pathogens. In addition to viscosin, Pf.SBW25 produces other metabolites with activity against plant pathogens. The production of these yet unknown metabolites appeared to be regulated by the GacS/GacA two-component regulatory system (the Gac-system). The second P. fluorescens strain SS101 (Pf.SS101) studied in this thesis is known for its plant growth-promoting activities but the underlying mechanisms and genes are largely unknown. Therefore, in this study, we aimed to identify novel metabolites and biosynthetic genes in Pf.SBW25 and Pf.SS101, and to investigate their role in plant growth promotion and biocontrol. To this end, a multidisciplinary approach involving bioinformatic analysis of the genome sequences of strains Pf.SBW25 and Pf.SS101, microarray-based expression profiling, screening of genomic libraries, bioactivity assays, mass spectrometric image analysis (MALDI-IMS) and GC/MSMS analysis was adopted. In conclusion, we showed that the GacS/GacA two-component system as a global regulator of the expression of genes play important roles in antagonism of Pseudomonas fluorescens toward plant pathogenic microbes as well as in plant growth promotion and ISR. Growth promotion by P. fluorescens is associated with alterations in auxin biosynthesis and transport, steroid biosynthesis, carbohydrate metabolism and sulfur assimilation. Moreover, advanced chemical profiling allowed us to compare the metabolite profiles of free-living P. fluorescens and P. fluorescens living in association with plant roots. A better understanding of yet unknown mechanisms exploited by the various Pseudomonas fluorescens strains will lead to new opportunities for the discovery and application of natural bioactive compounds for both industrial and agricultural purposes.

Thermococcus kodakarensis : the key to affordable biohydrogen production
Spaans, S.K. - \ 2016
University. Promotor(en): John van der Oost, co-promotor(en): Servé Kengen. - Wageningen : Wageningen University - ISBN 9789462577725 - 245 p.
thermococcus - thermococcus kodakarensis - hydrogen - bioenergy - canonical analysis - biosynthesis - nadph - archaea - microbiology - waterstof - bio-energie - canonische analyse - biosynthese - microbiologie
The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171 p.
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - biosynthese - peroxisomen - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - serine proteïnasen

Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.

A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.

As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.

To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.

Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.

Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.

The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.

Metabolic engineering of biosynthesis and sequestration of artemisinin
Wang, B. - \ 2016
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789462576728 - 210 p.
artemisinin - nicotiana benthamiana - arabidopsis - biosynthesis - malaria - drugs - genetic engineering - metabolism - artemisinine - biosynthese - geneesmiddelen - genetische modificatie - metabolisme

The sesquiterpenoid artemisinin (AN) is the most important medicine for the treatment of malaria in humans. The industrial production of AN still mainly depends on extraction from the plant Artemisia annua. However, the concentration of AN in A. annua is low. Although different engineering strategies have been used in both A. annua and heterologous plant and yeast production platforms, the worldwide capacity and production costs for AN are not in balance with its demand (Chapter 1). Although the genes encoding for the entire AN biosynthesis pathway (AN-PW) of the AN precursor dihydroartemisinic acid (DHAA) have been identified, the application of these genes in pathway engineering seem to be limited by lack of control over product transport and sequestration. At the onset of this thesis project there was no information on transport in the AN-PW. However, it was known that DHAA is converted into AN outside the glandular trichome cells of A. annua. Therefore, in this thesis I tried to gain more knowledge on transport within the AN-PW and the use of different metabolic engineering strategies to improve the production of AN.

At the onset of my PhD project, the AN-PW genes from two different A. annua chemotypes were compared to understand the basis of different relative activities in the two branches of the AN-PW (Chapter 2). For these assays we used transient expression in N. benthamiana. In the AN-PW, artemisinic aldehyde (AAA) is at a branch point as it can be converted to artemisinic acid (AA) by amorphadiene oxidase (AMO), or to dehydroartemisinic aldehyde (DHAAA) by artemisinic aldehyde Δ11 (13) reductase (DBR2). AA is the precursor for arteannuin B (AB) while DHAAA may be converted by a CYP71AV1 or an ALDH1 to dehydroartemisinic acid (DHAA), the precursor for AN. In this chapter we demonstrate that the CYP71AV1 from a high AN production (HAP) chemotype has reduced activity in the AB branch of the pathway compared to the CYP71AV1 from a low AN production (LAP) chemotype. In addition, we show that the relative expression levels of DBR2 and ALDH1 also affect the AN/AB chemotype. The low catalytic efficiency of AMO from the HAP chemotype may be caused by a deletion of seven amino acids at the N-terminus of the protein compared to CYP71AV1 from LAP. Ectopic expression of the AN-PW genes in N. benthamiana showed that the bulk of the PW products are modified by glycosylation and glutathione conjugations. These side reactions therefore compete with the biosynthesis flux towards the AN precursor DHAA. At this point in my thesis the ectopic expression of AN-PW genes in N. benthamiana had not yielded any AN. At a later stage it became clear that this was due to harvest of leaves at 5-7 days post agro-infiltration (dpi), while AN in N. benthamiana leaves expressing AN-PW genes only becomes detectable after 7 dpi.

Glycosylation of the bulk of the AN-PW products in N. benthamiana stresses the need for an efficient transport of (DH)AA to the outside of cells in order to escape from the glycosylation reactions. In Chapter 3, transport and sequestration of AN precursors was investigated by studying the effect of membrane transporters (PDRs) and lipid transfer proteins (LTPs). Hereto, two membrane transporters with activity towards AN-PW products were made available by the group of Prof. Marc Boutry and we isolated three LTP genes from Artemisia annua which showed expression in the glandular trichomes. In this chapter we show that AaLTP3 displays specific activity, together with AaPDR2 towards transport of (DH)AA to the apoplast in N. benthamiana. Moreover, infiltration experiments with (DH)AA in N. benthamiana leaves revealed that these compounds are rapidly taken up by the cells and that inside the cells there is a strong reverse flux in the AN-PW by conversion of (DH)AA towards (DH)AAA and (DH)AAOH. Subsequently we demonstrated that AaLTP3 has a stronger activity in keeping products in the apoplast than the AaPDR2 membrane transporter. Therefore, I suggest that by removal of (DH)AA from the cytosol through transport over the plasma membrane by AaPDR2 and subsequent sequestration in the apoplast by AaLTP3, AaLTP3 creates sink activity which prevents reflux of (DH)AA from the apoplast back into the cells. AaLTP3 therefore contributes to a directional flux through the AN-PW towards the end product (DH)AA. Finally, in this work we could also for the first time detect AN and AB in N. benthamiana leaves by extraction of necrotic leaves at 13 dpi.

Because in A. annua glandular trichome cells both the AN sesquiterpene biosynthesis pathway and the flavonoid biosynthesis pathway are active, we explored whether there is a functional interaction between these two major secondary metabolite biosynthesis pathways. In Chapter 4 we describe how we manipulate the flavonoid biosynthesis pathway in N. benthamiana leaves using the Antirhinum majus transcription factor Rosea1 (ROS) and test coexpression of ROS with AN-PW genes. The co-expression of ROS stimulates AN-PW product accumulation. Subsequent analysis indicates that this is most likely from transcriptional activation of the enzyme Mevalonate Kinase (MVK) in the mevalonate pathway, which provides precursors for the sesquiterpene biosynthesis pathway. In addition, we demonstrate that production of flavonoids competes with AN-PW product accumulation, as co-expression of AN-PW genes with ROS, but simultaneous inhibition of chalcone synthase (CHS) by a CHSRNAi construct, results in higher AN-PW product levels. However, accumulation of the end products AN and AB was not affected significantly. Finally, the combined expression of AN-PW+ROS+AaPDR2+AaLTP3+ CHSRNAi results in highest sequestration of (DH)AA in the apoplast and highest accumulation of the end products AN and AB in N. benthamiana.

During my thesis work, in a related project it was found that expression of another sesquiterpene biosynthesis gene (caryophyllene synthase; CST) in transgenic Arabidopsis resulted in higher caryophyllene emission for a transformant expressing a genomic DNA of CST, compared with a similar transformant expressing a CST cDNA described in literature. This suggested that ectopic expression of intron containing genes is more efficient than ectopic expression of cDNAs. To test whether in the context of metabolic engineering the use of genomic (intron-containing) genes is more efficient than the use of the corresponding cDNA we generated a set of stable transformed Arabidopsis lines with either genomic CST (gCST), cDNA CST (cCST), genomic amorphadiene synthesis (gADS) and cDNA ADS (cADS). In chapter 5 we show that indeed the lines with overexpression of the genomic clones yield higher levels of the anticipated products (caryophyllene or amorphadiene) than the lines with overexpression of the corresponding cDNAs. Transcript analysis showed that for gCST the increase in caryophyllene production was higher than can be explained solely by the increase in CST transcription. In the context of transient expression in N. benthamiana leaves the intron-mediated-enhancement effect was less pronounced.

In the final discussion chapter 6 I review limitations and potential solutions to metabolic engineering of the AN-PW in plants, and I discuss the impact of our findings on AN production capacity using transient expression versus natural production in A. annua. Moreover, I discuss how the finding of this thesis go beyond just insights into the AN-PW as especially the identification of the role of LTPs in sesquestration of (sesqui)terpenes into the apoplast may have an impact on the metabolic engineering efforts of many other (sesqui)terpene pathways. Because some plant hormones are also terpenoid products the newly identified role of LTPs may also have impact on a deeper understanding of hormone signalling in plants. I have already started exploring this path by generating a set of Arabidopsis plants with overexpression of different Arabidopsis LTP genes to test whether any hormone related traits are altered (Chapter 6). Preliminary results do indeed confirm a role of LTPs in endogenous plant hormone balance, something worthwhile to be further explored in future research.

The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. - \ 2015
University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
fungi contain very large numbers of genes related to SM production. Yet these genes are typically
silent under laboratory conditions. There are now many tools and strategies available to activate
or clone silent SM genes. This thesis details our efforts to apply various methods to define and
then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
containing many silent SM genes and gene clusters.

In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
of growth conditions. Heterologous expression of silent SM genes in a production host is also
discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
source of novel biosynthetic enzymes.

In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
of these SM genes or gene clusters were speculated, based on their similarity to those characterized
in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
fungal species. Remarkably, two predicted functional gene clusters were linked to the production
of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
findings, the majority of SM genes remained silent and only cladofulvin was detected. During
growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
another mechanism associated with a biotrophic lifestyle.

In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
of discovering new compounds. Many Ascomycete-specific global regulators of SM production
and morphological development in other fungi were identified in C. fulvum. We investigated
three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
This suggests that cladofulvin production is not required for asexual reproduction. The main
finding of this chapter is that global regulator manipulation cannot considered to be a universal
tool to discover new fungal natural products.

In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
is produced by many fungi and plants. Once synonymous only with constipation, this former
laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
forming novel dimers from existing monomers.

In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
active during early and late stages of infection of tomato, but are down-regulated during biotrophic
growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
inoculation (dpi). At each time point the infections were inspected microscopically to detect any
phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
(UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
results suggested that cladofulvin confers resistance to abiotic stress.

In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
enable discovery of homologous genes encoding enzymes with different specificities. Combining
bioinformatic and functional analyses should prove to be a powerful strategy for discovering
compounds with new biological activities, or enzymes relevant to metabolic engineering.

Diversity of aquatic Pseudomonas species and their activity against the fish pathogenic oomycete Saprolegnia
Liu, Y. ; Rzeszutek, E. ; Voort, M. van der; Wu, C.H. ; Thoen, E. ; Skaar, I. ; Bulone, V. ; Dorrestein, P.C. ; Raaijmakers, J.M. ; Bruijn, I. de - \ 2015
PLoS One 10 (2015)8. - ISSN 1932-6203 - 17 p.
cyclic lipopeptide surfactants - anguilla-australis richardson - media strain a199 - fluorescens - biosynthesis - parasitica - bacteria - chytridiomycosis - aquaculture - infections
Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.
Molecular and chemical dialogues in bacteria-protozoa interactions
Song, C. ; Mazzola, M. ; Cheng, X. ; Oetjen, J. ; Alexandrov, T. ; Dorrestein, P. ; Watrous, J. ; Voort, M. van der; Raaijmakers, J.M. - \ 2015
Scientific Reports 5 (2015). - ISSN 2045-2322 - 13 p.
imaging mass-spectrometry - pseudomonas-fluorescens cha0 - dictyostelium-discoideum - caenorhabditis-elegans - spatial segmentation - tetr family - aeruginosa - biosynthesis - rhizosphere - strain
Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.
Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels
Rombouts, W.H. ; Kort, D.W. de; Pham, T.T.H. ; Mierlo, C.P.M. van; Werten, M.W.T. ; Wolf, F.A. de; Gucht, J. van der - \ 2015
Biomacromolecules 16 (2015)8. - ISSN 1525-7797 - p. 2506 - 2513.
block-copolymers - gels - ph - gelatin - elastin - biosynthesis - proteins - polymers
Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.
High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose-binding protein as a solubility tag.
Huijbers, M.M.E. ; Berkel, W.J.H. van - \ 2015
Biotechnology Journal 10 (2015)3. - ISSN 1860-6768 - p. 395 - 403.
multifunctional puta flavoprotein - escherichia-coli - purification - domain - stress - biosynthesis - oxidase - crystallization - overexpression - identification
Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to ¿1-pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.
Isoprene emission by poplar is not important for the feeding behaviour of poplar leaf beetles
Müller, A. ; Kaling, M. ; Faubert, P. ; Gort, G. ; Smid, H.M. ; Loon, J.J.A. van; Dicke, M. ; Kanawati, B. ; Schmitt-Kopplin, P. ; Polle, A. ; Schnitzler, J.P. ; Rosenkranz, M. - \ 2015
BMC Plant Biology 15 (2015)1. - ISSN 1471-2229 - 16 p.
organic-compound emissions - chrysomela-populi - phratora-vitellinae - plant interactions - emitting poplars - volatiles - biosynthesis - caterpillars - performance - trichocarpa
Background Chrysomela populi (poplar leaf beetle) is a common herbivore in poplar plantations whose infestation causes major economic losses. Because plant volatiles act as infochemicals, we tested whether isoprene, the main volatile organic compound (VOC) produced by poplars (Populus x canescens), affects the performance of C. populi employing isoprene emitting (IE) and transgenic isoprene non-emitting (NE) plants. Our hypothesis was that isoprene is sensed and affects beetle orientation or that the lack of isoprene affects plant VOC profiles and metabolome with consequences for C. populi feeding. Results Electroantennographic analysis revealed that C. populi can detect higher terpenes, but not isoprene. In accordance to the inability to detect isoprene, C. populi showed no clear preference for IE or NE poplar genotypes in the choice experiments, however, the beetles consumed a little bit less leaf mass and laid fewer eggs on NE poplar trees in field experiments. Slight differences in the profiles of volatile terpenoids between IE and NE genotypes were detected by gas chromatography - mass spectrometry. Non-targeted metabolomics analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometer revealed genotype-, time- and herbivore feeding-dependent metabolic changes both in the infested and adjacent undamaged leaves under field conditions. Conclusions We show for the first time that C. populi is unable to sense isoprene. The detected minor differences in insect feeding in choice experiments and field bioassays may be related to the revealed changes in leaf volatile emission and metabolite composition between the IE and NE poplars. Overall our results indicate that lacking isoprene emission is of minor importance for C. populi herbivory under natural conditions, and that the lack of isoprene is not expected to change the economic losses in poplar plantations caused by C. populi infestation.
Drought stress affects plant metabolites and herbivore preference but not host location by its parasitoids
Weldegergis, B.T. ; Zhu, F. ; Poelman, E.H. ; Dicke, M. - \ 2015
Oecologia 177 (2015)3. - ISSN 0029-8549 - p. 701 - 713.
volatile emissions - water-stress - abiotic factors - oviposition - genes - biosynthesis - consequences - lepidoptera - complexity - expression
One of the main abiotic stresses that strongly affects plant survival and the primary cause of crop loss around the world is drought. Drought stress leads to sequential morphological, physiological, biochemical and molecular changes that can have severe effects on plant growth, development and productivity. As a consequence of these changes, the interaction between plants and insects can be altered. Using cultivated Brassica oleracea plants, the parasitoid Microplitis mediator and its herbivorous host Mamestra brassicae, we studied the effect of drought stress on (1) the emission of plant volatile organic compounds (VOCs), (2) plant hormone titres, (3) preference and performance of the herbivore, and (4) preference of the parasitoid. Higher levels of jasmonic acid (JA) and abscisic acid (ABA) were recorded in response to herbivory, but no significant differences were observed for salicylic acid (SA) and indole-3-acetic acid (IAA). Drought significantly impacted SA level and showed a significant interactive effect with herbivory for IAA levels. A total of 55 VOCs were recorded and the difference among the treatments was influenced largely by herbivory, where the emission rate of fatty acid-derived volatiles, nitriles and (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT] was enhanced. Mamestra brassicae moths preferred to lay eggs on drought-stressed over control plants; their offspring performed similarly on plants of both treatments. VOCs due to drought did not affect the choice of M. mediator parasitoids. Overall, our study reveals an influence of drought on plant chemistry and insect-plant interactions.
Regulation and natural functions of lipopeptide biosynthesis in Pseudomonas
Song, C. - \ 2015
University. Promotor(en): Francine Govers, co-promotor(en): Jos Raaijmakers. - Wageningen : Wageningen University - ISBN 9789462572690 - 173
pseudomonas fluorescens - lipoproteïnen - biosynthese - genetische kartering - genregulatie - genomica - transcriptomica - verdedigingsmechanismen - protozoa - mutanten - lipoproteins - biosynthesis - genetic mapping - gene regulation - genomics - transcriptomics - defence mechanisms - mutants

Summary

Lipopeptides (LPs) are surface-active, antimicrobial compounds composed of a lipid moiety linked to a short linear or cyclic oligopeptide. In bacteria, LPs are synthesized by large nonribosomal peptide synthetases (NRPSs) via a thiotemplate process. Compared to the understanding of LP biosynthesis, little is known about the genetic regulation.

The aims of this PhD thesis were to identify new regulatory genes of LP biosynthesis and to unravel the natural functions of LPs in plant-associated Pseudomonas species. Using a combination of various ‘omics’-based technologies, we identified two small RNAs, designated RsmY and RsmZ, that, together with the repressor proteins RsmA and RsmE, regulate the biosynthesis of the LP massetolide in the rhizosphere bacterium Pseudomonas fluorescens SS101. Four other regulatory genes (phgdh, dnaK, prtR and clpA) of massetolide biosynthesis were identified via random mutagenesis. Mutations in each of these four genes caused a deficiency in massetolide production, swarming motility and biofilm formation, two natural functions associated with the production of LPs in Pseudomonas. Results further indicated that the ClpAP protease complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.

LPs exhibit broad-spectrum antimicrobial activities and have diverse natural functions for the producing bacteria. LPs of P. fluorescens were shown to play an important role in defense against protozoan predation. Genome-wide transcriptome analysis revealed that 55 and 73 genes were up- and down-regulated respectively in P. fluorescens strain SS101 upon grazing by the protozoan predator Naeglaria americana. The up-regulated genes included the LP biosynthesis genes massABC, but also genes involved in alkane degradation and in putrescine catalysis. Putrescine induced encystment of the protozoa, possibly providing a second line of defense against predation. MALDI imaging mass spectrometry (IMS) and live colony NanoDesi mass spectrometry further revealed, in real time, site-specific LP production at the interface of Pseudomonas-protozoa interactions. When the closely related strain P. fluorescens SBW25 was exposed to N. americana, similar overall transcriptional and metabolic responses were observed as found for strain SS101, but also strain-specific responses were apparent. These results indicate that closely related bacterial strains exhibit common and unique transcriptomic and metabolic responses to protozoan predation. Next to defense against competitors and predators, LPs are well-known for their role in swarming motility, a flagella-driven multicellular behavior of bacteria. Orfamide-deficient mutants of P. protegens Pf-5, either with deletions in the biosynthesis gene ofaA or in the regulatory gene gacA, cannot swarm on their own but ‘hitch-hike’ with parental strain Pf-5. However, distinctly different spatial distributions in co-swarming colonies were observed for these two mutants, with the ofaA mutant moving behind the wild type and the gacA mutant predominating on the edge of the swarming colony. Subsequent experimental evolution assays showed that repeated swarming cycles of strain Pf-5 drives parallel evolution toward fixation of spontaneous gacS/gacA mutants on the edge, ultimately causing colony collapse. Transcriptome analyses revealed that genes associated with resource acquisition, motility, chemotaxis and efflux were significantly upregulated in these regulatory mutants. Moreover, microscopic analysis showed that gacA mutant cells were longer and more flagellated than wild type and ofaA mutant cells, which may explain their predominance on the edge of co-swarming colonies. Collectively, these results indicated that adaptive convergent evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these spontaneous mutations during dispersal of bacteria into new territories are frequency-dependent.

Bridging domains : a comparison between information processing in Archaea and Eukarya
Koning, B. de - \ 2015
University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - Wageningen : Wageningen University - ISBN 9789462572379 - 154
archaea - transfer rna - rna-polymerase - transcriptieregulatie - biosynthese - sulfolobus solfataricus - rna polymerase - regulation of transcription - biosynthesis

Bridging Domains

A Comparison between Information Processing in Archaea and Eukarya

Studying Information Processing

Living cells evolved complex systems to handle the flow of information both accurately and efficiently. These systems are highly comparable between the three domains of life: eukaryotes, bacteria and archaea. The central components of replication, transcription, aminoacylation, and translation are found in every living cell known today, with only relatively small deviations, despite a separation of billions of years of evolution. Archaea are unicellular, do not contain organelles, and have relatively small genomes, so are, at first sight, quite similar to their far better known prokaryotic cousins: the bacteria. Nevertheless, if it comes down to information processing, archaea are, surprisingly, more related to eukaryotes than to bacteria, both at the sequence level of RNA and proteins, and at the architecture level of key complexes as well. This makes them excellent model systems to study eukaryote-like information processing. The absence of cell specialization, less cell organization, less or even no intracellular compartmentalization, and less intensive regulation, have proven to give a clearer picture of the function of conserved key elements within these complex systems. [Chapter 2]

In this thesis, we report several attempts to elucidate functional details of some very conserved factors in information processing in S. solfataricus using recently established genetic modification techniques. S. solfataricus is a thermoacidophilic crenarchaeote that grows optimally at temperatures between 70°C and 85°C and at pH values between 2 and 3. Its genome sequence is known since 2001. Best practices have become standardized between laboratories, and the genomic toolbox includes gene knockout, overexpression systems, the availability of reporter genes, and tunable promoters.

MBF1, a highly conserved activator

MBF1 (multi-protein bridging factor 1) is reported to be a transcriptional co-activator in eukaryotes. It was shown to cross the gap between transcription regulators and the transcriptional machinery itself. MBF1 was found to be highly conserved within archaea, being present in almost all species with the key exception of marine thaumarchaeotes. However, none of the associated transcription regulators were known to be present within the archaeal domain, raising the question whether a class of other regulators was overlooked, or that archaeal MBF1 might be a transcriptional activator itself, binding to DNA directly instead of indirectly via a binding partner. Additionally one study revealed a surprising dual role of this protein: in yeast it was not only associated with transcription but contributed to translation fidelity as well. A neighbourhood analysis across the archaeal domain revealed no clear preference for either transcription or translation. Elements of both systems are equally present, especially in the well conserved neighbourhood within the crenarchaeotes. [Chapter 3]

A mbf1 disruption mutant of the S. solfataricus was made using heterozygous recombination with a suicide plasmid. Under standard laboratory growth conditions mbf1 appears to be not essential for growth, and comparing growth characteristics with its parental strain did not reveal striking differences between the two. It was observed, that the Sulfolobus mbf1 disruption mutant is much more sensitive during cultivation than its parental strain, showing sudden death during growth much more often. Being hard to quantify, this behaviour was especially observed when cultures were transferred at later stages during stationary phase or unfrozen from long term storage. But the largest difference was observed in the increased sensitivity of the mbf1 disruption mutant towards paromomycin. Paromomycin is an aminoglycoside-type antibiotic that interferes with the recognition of cognate codon-anti-codon binding within the ribosomes during translation. [Chapter 4]

A more detailed study to the molecular characteristics of the archaeal MBF1 from S. solfataricus revealed hardly any associations to the transcription machinery, but strengthened the assumed association to the translation apparatus. It was found that archaeal MBF1 consists of two domains that are structurally independent: an N-terminal zinc-ribbon, which is not conserved beyond the archaeal MBF1s, and the well conserved C-terminal HTH-domain (helix-turn-helix domain). This C-terminal HTH domain was shown to bind to the small ribosomal subunit by affinity purification, and in co-purification experiments, in which we detected the presence of archaeal MBF1 in ribosomal purifications. NMR structure comparisons confirmed that archaeal MBF1 binds to the small ribosomal subunit using its C-terminal HTH domain, whereas the N-terminal zinc-ribbon might only contribute to this interaction, but does not participate directly in binding. [Chapter 5]

Altogether, these findings made us believe that MBF1s in archaea are not associated with transcription but rather with translation. Based on the observations in yeast, and more recently its binding to polyadenylated mRNAs in different eukaryotic species, and, against the backdrop that the protein domain that binds to the small ribosomal subunit in S. solfataricus is highly conserved across the archaeao-eukaryotic lineage, it is tempting to speculate that the eukaryotic MBF1 plays a comparable role in the translation process in eukaryotes as well.

TGT, a conserved dichotomy

Another well conserved element within all three domains of life, which is involved in information processing, is the TGT (tRNA-guanine transglycosylase) family of proteins. This family of proteins shows a clear dichotomy: TGT is responsible for the exchange of guanine at the wobble position (position 34) of the anti-codon of certain tRNAs with either queuosine in eukaryotes or its precursor preQ1 in bacteria, whereas, in archaea, TGT is responsible for the exchange of guanine with preQ0 at position 15 in almost, if not all, archaeal tRNAs. PreQ0 is in a later stage converted to archaeosine by another protein that belongs to the TGT family as well.

Disruption of the tgt gene, which encodes the TGT protein in S. solfataricus, revealed that it was solely responsible for this process without any redundancy present. Like mbf1, this gene appeared to be non-essential, as this mutant was also as viable as its parental strain, and showed hardly any changes in growth characteristics. In comparison to the mbf1 disruption mutant, the tgt disruption mutant was much more stable and did not reveal the sensitivity to stationary phase. It grew slightly slower than the parental strain, especially at normal temperatures (75°C), but when temperature levels were raised (87-93°C) growth returned to almost wild-type levels. [Chapter 6]

Aiding research to the basal machinery of RNAP

Beyond doubt, the best studied, element of information processing systems is the RNAP (RNA polymerase) complex. Its basal core is present in all known life forms, and is highly conserved. The surrounding, auxiliary, and regulatory elements are less conserved, but, nevertheless, the archaeal RNAP is almost identical to the eukaryotic RNAP II complex (see figure). This high resemblance already proved beneficial, as the heterologous expression of the archaeal RNAP revealed numerous functional details about the molecular characteristics of the complex as a whole, and, in addition, revealed also an unprecedented insight in the separate subunits as this provided opportunities to tamper with the subunit composition and to modify the separate subunits themselves by introducing genetic variations.

Unfortunately, purification of homologously expressed complexes, which are expressed in archaeal systems itself, are, in contrast to ones heterologously expressed in bacterial hosts, hard to obtain, and involve a number of purification steps and therefore a substantial amount of biomass. To enable easier purification, a method was developed in which a purification tag was inserted in the genome of S. solfataricus after a gene that encodes an RNAP subunit, avoiding artificial overproduction by viral infections or heterologous expression in other less adapted hosts. In a proof of principle experiment, the enrichment an RNAP core component was proven, whereas an auxiliary element was tagged using this novel method. [Chapter 7]

Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity
Yang, T. ; Gao, L. ; Hu, H. ; Stoopen, G.M. ; Wang, C. ; Jongsma, M.A. - \ 2014
Journal of Biological Chemistry 289 (2014). - ISSN 0021-9258 - p. 36325 - 36335.
dimethylallyl diphosphate - squalene synthase - monoterpene synthase - terpene synthases - biosynthesis - expression - arabidopsis - metabolism - cloning - leaves
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteinsare known to catalyzethis cyclopropanation reactionof terpene precursors.Two of them, phytoene and squalenesynthase, arebifunctional enzymes with both prenyltransferase and terpenesynthase activity. CDS, the other member,was reported to perform only the prenyltransferase step.Here, we show thatthe NDXXD catalytic motif of CDS,under lowersubstrate conditions prevalent inplants,also catalyzesthe next step converting CPP into chrysanthemolby hydrolyzing the diphosphatemoiety.The enzymatic hydrolysis reactionfollowed conventional Michaelis-Menten kinetics, withaKM value for CPP of 196 µM.For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemolwas ~100 µM, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemolat arate of 0.12 –0.16 µg•h-1•g-1FW. We propose that CDS should be renamed a chrysanthemol synthase(CHS)utilizingDMAPP as substrate.
Diversity of pyrrolizidine alkaloids in native and invasive Senecio pterophorus (Asteraceae): Implications for toxicity
Castells, E. ; Mulder, P.P.J. ; Perez -Trujillo, M. - \ 2014
Phytochemistry 108 (2014). - ISSN 0031-9422 - p. 137 - 146.
increased competitive ability - mass-spectrometry - enemy release - chemical diversity - hypothesis - plants - biosynthesis - evolution - metabolism - defense
Changes in plant chemical defenses after invasion could have consequences on the invaded ecosystems by modifying the interactions between plants and herbivores and facilitating invasion success. However, no comprehensive biogeographical studies have yet determined the phenotypic levels of plant chemical defenses, as consumed by local herbivores, covering large distributional areas of a species. Senecio pterophorus is a perennial shrub native to Eastern South Africa, expanded into Western South Africa and introduced into Australia and Europe. As other Asteraceae, S. pterophorus contains pyrrolizidine alkaloids (PAs) toxic to vertebrate and invertebrate herbivores. Here we analyzed S. pterophorus PAs by LC–MS/MS on foliage sampled across its entire distributional range, including the native and all non-native areas. PA concentrations and diversity was very high: we found 57 compounds belonging to 6 distinct necine base-types, including the highly toxic 1,2-unsaturated PAs (retronecine and otonecines) and the less toxic 1,2-saturated PAs (platynecine and rosmarinecines). Plants from different origins diverged in their PA absolute and relative concentrations. Rosmarinine was the most abundant compound in Australia and South Africa, but it was nearly absent in Europe. We characterized three plant chemotypes: retrorsine–senkirkine chemotype in Eastern South Africa, rosmarinine chemotype in Australia and Western South Africa, and acetylseneciphylline chemotype in Europe. PA absolute concentrations were highest in Australia. The increased absolute and relative concentrations of retronecine PAs from Australia and Europe, respectively, indicate that S. pterophorus is potentially more toxic in the invasive range than in the native range.
An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato
Gomez Roldan, M.V. ; Outchkourov, N.S. ; Houwelingen, A.M.M.L. van; Lammers, M. ; Romero Fuente, I. ; Ziklo, N. ; Aharoni, A. ; Hall, R.D. ; Beekwilder, M.J. - \ 2014
The Plant Journal 80 (2014)4. - ISSN 0960-7412 - p. 695 - 708.
plant transformation - transcription factor - petunia-hybrida - fruit - biosynthesis - expression - protein - system - metabolome - infection
Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.
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