Publications by authors named "Jules Beekwilder"

103 Publications

Integrating structure-based machine learning and co-evolution to investigate specificity in plant sesquiterpene synthases.

PLoS Comput Biol 2021 Mar 22;17(3):e1008197. Epub 2021 Mar 22.

Bioinformatics Group, Department of Plant Sciences, Wageningen University and Research, Wageningen, The Netherlands.

Sesquiterpene synthases (STSs) catalyze the formation of a large class of plant volatiles called sesquiterpenes. While thousands of putative STS sequences from diverse plant species are available, only a small number of them have been functionally characterized. Sequence identity-based screening for desired enzymes, often used in biotechnological applications, is difficult to apply here as STS sequence similarity is strongly affected by species. This calls for more sophisticated computational methods for functionality prediction. We investigate the specificity of precursor cation formation in these elusive enzymes. By inspecting multi-product STSs, we demonstrate that STSs have a strong selectivity towards one precursor cation. We use a machine learning approach combining sequence and structure information to accurately predict precursor cation specificity for STSs across all plant species. We combine this with a co-evolutionary analysis on the wealth of uncharacterized putative STS sequences, to pinpoint residues and distant functional contacts influencing cation formation and reaction pathway selection. These structural factors can be used to predict and engineer enzymes with specific functions, as we demonstrate by predicting and characterizing two novel STSs from Citrus bergamia.
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http://dx.doi.org/10.1371/journal.pcbi.1008197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016262PMC
March 2021

Several geranylgeranyl diphosphate synthase isoforms supply metabolic substrates for carotenoid biosynthesis in tomato.

New Phytol 2021 Feb 16. Epub 2021 Feb 16.

Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, Barcelona, 08193, Spain.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms. The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.
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http://dx.doi.org/10.1111/nph.17283DOI Listing
February 2021

The application of a biostimulant based on tannins affects root architecture and improves tolerance to salinity in tomato plants.

Sci Rep 2021 Jan 11;11(1):354. Epub 2021 Jan 11.

Plant Physiology Unit, Department of Life Sciences and Systems Biology, University of Torino, Turin, Italy.

Roots have important roles for plants to withstand adverse environmental conditions, including salt stress. Biostimulant application was shown to enhance plant resilience towards abiotic stresses. Here, we studied the effect of a tannin-based biostimulant on tomato (Solanum lycopersicum L.) grown under salt stress conditions. We investigated the related changes at both root architecture (via imaging and biometric analysis) and gene expression (RNA-Seq/qPCR) levels. Moreover, in order to identify the main compounds potentially involved in the observed effects, the chemical composition of the biostimulant was evaluated by UV/Vis and HPLC-ESI-Orbitrap analysis. Sixteen compounds, known to be involved in root development and having a potential antioxidant properties were identified. Significant increase of root weight (+ 24%) and length (+ 23%) was observed when the plants were grown under salt stress and treated with the biostimulant. Moreover, transcriptome analysis revealed that the application of the biostimulant upregulated 285 genes, most of which correlated to root development and salt stress tolerance. The 171 downregulated genes were mainly involved in nutrient uptake. These data demonstrated that the biostimulant is able not only to restore root growth in salty soils, but also to provide the adequate plant nourishment by regulating the expression of essential transcription factors and stress responsive genes.
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http://dx.doi.org/10.1038/s41598-020-79770-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801735PMC
January 2021

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Arch Biochem Biophys 2020 11 26;695:108647. Epub 2020 Oct 26.

Bioscience, Wageningen Plant Research, Netherlands. Electronic address:

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.
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http://dx.doi.org/10.1016/j.abb.2020.108647DOI Listing
November 2020

The transition of Rhodobacter sphaeroides into a microbial cell factory.

Biotechnol Bioeng 2021 Feb 23;118(2):531-541. Epub 2020 Oct 23.

Bioprocess Engineering, Wageningen University, Wageningen, The Netherlands.

Microbial cell factories are the workhorses of industrial biotechnology and improving their performances can significantly optimize industrial bioprocesses. Microbial strain engineering is often employed for increasing the competitiveness of bio-based product synthesis over more classical petroleum-based synthesis. Recently, efforts for strain optimization have been standardized within the iterative concept of "design-build-test-learn" (DBTL). This approach has been successfully employed for the improvement of traditional cell factories like Escherichia coli and Saccharomyces cerevisiae. Within the past decade, several new-to-industry microorganisms have been investigated as novel cell factories, including the versatile α-proteobacterium Rhodobacter sphaeroides. Despite its history as a laboratory strain for fundamental studies, there is a growing interest in this bacterium for its ability to synthesize relevant compounds for the bioeconomy, such as isoprenoids, poly-β-hydroxybutyrate, and hydrogen. In this study, we reflect on the reasons for establishing R. sphaeroides as a cell factory from the perspective of the DBTL concept. Moreover, we discuss current and future opportunities for extending the use of this microorganism for the bio-based economy. We believe that applying the DBTL pipeline for R. sphaeroides will further strengthen its relevance as a microbial cell factory. Moreover, the proposed use of strain engineering via the DBTL approach may be extended to other microorganisms that have not been critically investigated yet for industrial applications.
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http://dx.doi.org/10.1002/bit.27593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894463PMC
February 2021

Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response.

Nat Genet 2020 10 28;52(10):1111-1121. Epub 2020 Sep 28.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.
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http://dx.doi.org/10.1038/s41588-020-0690-6DOI Listing
October 2020

MYB5-like and bHLH influence flavonoid composition in pomegranate.

Plant Sci 2020 Sep 20;298:110563. Epub 2020 Jun 20.

Consiglio Nazionale delle Ricerche, Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (CNR-ISAFOM) U.O.S. Catania, Via Empedocle, 58, 95128, Catania, Italy.

The fruit of the pomegranate (Punica granatum L.) is an important nutraceutical food rich in polyphenolic compounds, including hydrolysable tannins, anthocyanins and flavonols. Their composition varies according to cultivar, tissue and fruit development stage and is probably regulated by a combination of MYB and bHLH type transcription factors (TFs). In this study, metabolomics analysis during fruit developmental stages in the main pomegranate cultivars, Wonderful and Valenciana with contrasting colour of their ripe fruits, showed that flavonols were mostly present in flowers while catechins were highest in unripe fruits and anthocyanins in late fruit maturation stages. A novel MYB TF, PgMYB5-like, was identified, which differs from previously isolated pomegranate TFs by unique C-terminal protein motifs and lack of the amino-acid residues conserved among anthocyanins promoting MYBs. In both pomegranate cultivars the expression of PgMYB5-like was high at flowering stage, while it decreased during fruit ripening. A previously identified bHLH-type TF, PgbHLH, also showed high transcript levels at flowering stage in both cultivars, while it showed a decrease in expression during fruit ripening in cv. Valenciana, but not in cv. Wonderful. Functional analysis of both TFs was performed by agro-infiltration into Nicotiana benthamiana leaves. Plants infiltrated with the PgMYB5-like+PgbHLH combined construct showed a specific and significant accumulation of intermediates of the flavonoid pathway, especially dihydroflavonols, while anthocyanins were not produced. Thus, we propose a role for PgMYB5-like and PgbHLH in the first steps of flavonoid production in flowers and in unripe fruits. The expression patterns of these two TFs may be key in determining the differential flavonoid composition in both flowers and fruits of the pomegranate varieties Wonderful and Valenciana.
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http://dx.doi.org/10.1016/j.plantsci.2020.110563DOI Listing
September 2020

Growth-uncoupled isoprenoid synthesis in .

Biotechnol Biofuels 2020 13;13:123. Epub 2020 Jul 13.

Bioprocess Engineering, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Background: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.

Results: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.

Conclusions: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.
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http://dx.doi.org/10.1186/s13068-020-01765-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359475PMC
July 2020

A Biostimulant Seed Treatment Improved Heat Stress Tolerance During Cucumber Seed Germination by Acting on the Antioxidant System and Glyoxylate Cycle.

Front Plant Sci 2020 17;11:836. Epub 2020 Jun 17.

Plant Physiology Unit, Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.

Seed enhancement technologies have the potential to improve germination and seedling growth under environmental stress. The effects of KIEM, an innovative biostimulant based on lignin derivatives and containing plant-derived amino acids and molybdenum, were investigated on cucumber ( L.) seed germination. To determine the metabolic targets of this product, biometric, transcriptional and biochemical analyses were carried out on both non-treated and KIEM-treated seeds incubated for 24 and 48 h under standard (28°C) and heat stress (35°C) conditions. The application of the biostimulant as a seed treatment increased the percent germination (+6.54%) and fresh biomass (+13%) at 48 h, and decreased the content of HO in treated seeds at 28°C (-70%) and at 35°C (-80%). These changes in biometric and biochemical properties were accompanied by changes in expression levels of the genes coding for ROS-producing (RBOH) and scavenging (SOD, CAT, GST) enzymes and their specific activity. In general, the treatment with KIEM in heat-stress condition appeared to stimulate a higher accumulation of three scavenger gene transcripts: (+1.78), (+1.75), and (+3.39), while the isoform was dramatically downregulated (0.24). Moreover, the amount of non-protein thiols, important antioxidant molecules, was increased by the biostimulant after 48 h (+20%). Taken together these results suggest that KIEM acts through mitigation of the effects of the oxidative stress. Moreover, after 48 h, the pre-sowing treatment with KIEM increased the transcription levels (+1.5) and the activity of isocitrate lyase (+37%), a key enzyme of the glyoxylate cycle, suggesting a potential effect of this product in speeding up the germination process. Finally, the chemical characterization of KIEM identified five essential and three non-essential amino acids, and others bioactive compounds, including five organic and inorganic acids that might be potentially involved in its activity. Based on these data, insights on the potential mechanism of action of the biostimulant, suggested that there are broader applications as a product able to increase seed tolerance to different abiotic stress typical of adverse environmental conditions.
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http://dx.doi.org/10.3389/fpls.2020.00836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311796PMC
June 2020

Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories.

Trends Biotechnol 2020 08 13;38(8):917-934. Epub 2020 Apr 13.

Laboratory of Food Chemistry, Wageningen University & Research, Bornse Weilanden 9, 6708 WG, Wageningen, Netherlands. Electronic address:

In plants, prenylation of aromatic compounds, such as (iso)flavonoids and stilbenoids, by membrane-bound prenyltransferases (PTs), is an essential step in the biosynthesis of many bioactive compounds. Prenylated aromatic compounds have various health-beneficial properties that are interesting for industrial applications, but their exploitation is limited due to their low abundance in nature. Harnessing plant aromatic PTs for prenylation in microbial cell factories may be a sustainable and economically viable alternative. Limitations in prenylated aromatic compound production have been identified, including availability of prenyl donor substrate. In this review, we summarize the current knowledge about plant aromatic PTs and discuss promising strategies towards the optimized production of prenylated aromatic compounds by microbial cell factories.
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http://dx.doi.org/10.1016/j.tibtech.2020.02.006DOI Listing
August 2020

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

Microb Biotechnol 2020 07 24;13(4):1082-1093. Epub 2020 Mar 24.

Bioprocess Engineering, Wageningen University, 6708PB, Wageningen, The Netherlands.

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4- C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.
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http://dx.doi.org/10.1111/1751-7915.13562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264872PMC
July 2020

Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes.

J Exp Bot 2020 05;71(10):3052-3065

Wageningen Plant Research, 6700 AA, Wageningen, The Netherlands.

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.
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http://dx.doi.org/10.1093/jxb/eraa086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260718PMC
May 2020

Metabolic flux ratio analysis by parallel C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides.

Metab Eng 2020 01 13;57:228-238. Epub 2019 Dec 13.

Bioprocess Engineering, Wageningen University, 6708PB, Wageningen, the Netherlands. Electronic address:

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-C]- and [4-C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.
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http://dx.doi.org/10.1016/j.ymben.2019.12.004DOI Listing
January 2020

Silencing of germacrene A synthase genes reduces guaianolide oxalate content in L.

GM Crops Food 2020 31;11(1):54-66. Epub 2019 Oct 31.

Institute for Biological Research "Siniša Stanković"- National Institute of Republic of Serbia, University of Belgrade, Belgrade, Republic of Serbia.

Chicory ( L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by ) and GAS short (encoded by ). AmiRNA silencing was used to obtain plants with reduced gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.
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http://dx.doi.org/10.1080/21645698.2019.1681868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064209PMC
February 2020

Glucosinolate variability between turnip organs during development.

PLoS One 2019 6;14(6):e0217862. Epub 2019 Jun 6.

Wageningen Plant Research, Wageningen, The Netherlands.

Turnip (Brassica rapa spp. rapa) is an important vegetable species, with a unique physiology. Several plant parts, including both the turnip tubers and leaves, are important for human consumption. During the development of turnip plants, the leaves function as metabolic source tissues, while the tuber first functions as a sink, while later the tuber turns into a source for development of flowers and seeds. In the present study, chemical changes were determined for two genotypes with different genetic background, and included seedling, young leaves, mature leaves, tuber surface, tuber core, stalk, flower and seed tissues, at seven different time points during plant development. As a basis for understanding changes in glucosinolates during plant development, the profile of glucosinolates was analysed using liquid chromatography (LC) coupled to mass spectrometry (MS). This analysis was complemented by a gene expression analysis, focussed on GLS biosynthesis, which could explain part of the observed variation, pointing to important roles of specific gene orthologues for defining the chemical differences. Substantial differences in glucosinolate profiles were observed between above-ground tissues and turnip tuber, reflecting the differences in physiological role. In addition, differences between the two genotypes and between tissues that were harvested early or late during the plant lifecycle. The importance of the observed differences in glucosinolate profile for the ecophysiology of the turnip and for breeding turnips with optimal chemical profiles is discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217862PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553741PMC
February 2020

Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids.

J Agric Food Chem 2019 Dec 2;67(49):13478-13486. Epub 2019 May 2.

Laboratory of Food Chemistry , Wageningen University & Research , Bornse Weilanden 9 , 6708 WG Wageningen , Netherlands.

Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast . As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from in naringenin-producing yeast strains resulted in production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.
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http://dx.doi.org/10.1021/acs.jafc.9b01367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909231PMC
December 2019

A MYB Triad Controls Primary and Phenylpropanoid Metabolites for Pollen Coat Patterning.

Plant Physiol 2019 05 12;180(1):87-108. Epub 2019 Feb 12.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel

The pollen wall is a complex, durable structure essential for plant reproduction. A substantial portion of phenylpropanoids (e.g. flavonols) produced by pollen grain tapetal cells are deposited in the pollen wall. Transcriptional regulation of pollen wall formation has been studied extensively, and a specific regulatory mechanism for Arabidopsis () pollen flavonol biosynthesis has been postulated. Here, metabolome and transcriptome analyses of anthers from mutant and overexpression genotypes revealed that Arabidopsis MYB99, a putative ortholog of the petunia () floral scent regulator ODORANT1 (ODO1), controls the exclusive production of tapetum diglycosylated flavonols and hydroxycinnamic acid amides. We discovered that MYB99 acts in a regulatory triad with MYB21 and MYB24, orthologs of emission of benzenoids I and II, which together with ODO1 coregulate petunia scent biosynthesis genes. Furthermore, promoter-activation assays showed that MYB99 directs precursor supply from the Calvin cycle and oxidative pentose-phosphate pathway in primary metabolism to phenylpropanoid biosynthesis by controlling expression. We provide a model depicting the relationship between the Arabidopsis MYB triad and structural genes from primary and phenylpropanoid metabolism and compare this mechanism with petunia scent control. The discovery of orthologous protein triads producing related secondary metabolites suggests that analogous regulatory modules exist in other plants and act to regulate various branches of the intricate phenylpropanoid pathway.
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http://dx.doi.org/10.1104/pp.19.00009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501115PMC
May 2019

An analysis of characterized plant sesquiterpene synthases.

Phytochemistry 2019 Feb 13;158:157-165. Epub 2018 Nov 13.

Bioinformatics Group, Department of Plant Sciences, Wageningen University, Netherlands; Biometris, Department of Plant Sciences, Wageningen University, Netherlands. Electronic address:

Plants exhibit a vast array of sesquiterpenes, C15 hydrocarbons which often function as herbivore-repellents or pollinator-attractants. These in turn are produced by a diverse range of sesquiterpene synthases. A comprehensive analysis of these enzymes in terms of product specificity has been hampered by the lack of a centralized resource of sufficient functionally annotated sequence data. To address this, we have gathered 262 plant sesquiterpene synthase sequences with experimentally characterized products. The annotated enzyme sequences allowed for an analysis of terpene synthase motifs, leading to the extension of one motif and recognition of a variant of another. In addition, putative terpene synthase sequences were obtained from various resources and compared with the annotated sesquiterpene synthases. This analysis indicated regions of terpene synthase sequence space which so far are unexplored experimentally. Finally, we present a case describing mutational studies on residues altering product specificity, for which we analyzed conservation in our database. This demonstrates an application of our database in choosing likely-functional residues for mutagenesis studies aimed at understanding or changing sesquiterpene synthase product specificity.
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http://dx.doi.org/10.1016/j.phytochem.2018.10.020DOI Listing
February 2019

Identification of the Bisabolol Synthase in the Endangered Candeia Tree ( (DC) McLeisch).

Front Plant Sci 2018 19;9:1340. Epub 2018 Sep 19.

Wageningen Plant Research, Wageningen, Netherlands.

Candeia ( (DC) McLeisch, Asteraceae) is a Brazilian tree, mainly occurring in the cerrado areas. From ethnobotanical information its essential oil is known to have wound healing and nociceptive properties. These properties are ascribed to result from a sesquiterpene alcohol, (-)-α-bisabolol, which is present at high concentrations in this oil. Bisabolol is highly valued by the cosmetic industry because of its antibacterial, anti-inflammatory, skin-smoothing and wound healing properties. Over the past decades, Candeia timber has been collected at large scale for bisabolol extraction from wild reserves and the species is thereby at risk of extinction. To support the development of breeding and nursing practices that would facilitate sustainable cultivation of Candeia, we identified a terpene synthase gene, EeBOS1, that appears to control biosynthesis (-)-α-bisabolol in the plant. Expression of this gene in showed that EeBOS1 protein is capable of producing (-)-α-bisabolol from farnesyl pyrophosphate . Analysis of gene expression in different tissues from Candeia plants in different life stages showed a high correlation of EeBOS1 expression and accumulation of (-)-α-bisabolol. This work is the first step to unravel the pathway toward (-)-α-bisabolol in Candeia, and in the further study of the control of (-)-α-bisabolol production.
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http://dx.doi.org/10.3389/fpls.2018.01340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158398PMC
September 2018

In depth investigation of the metabolism of Nectandra megapotamica chemotypes.

PLoS One 2018 6;13(8):e0201996. Epub 2018 Aug 6.

Laboratório de Produtos Naturais e Espectrometria de Massas, Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brasil.

Plants produce a wide range of secondary metabolites. Within a single species, chemotypes can be distinguished by the differences in the composition of the secondary metabolites. Herein, we evaluated Nectandra megapotamica (Spreng.) chemotypes and the balance of different classes of metabolites to verify how significant differences in plant metabolism are regarding chemotypes. We collected N. megapotamica leaves from eight adult plants in two Brazilian states. The essential oils and ethanol/water extracts were analyzed by GC-MS and LC-DAD-MS, respectively. Histochemical tests were performed, as well as chemical analyses of leaves from adaxial and abaxial foliar surfaces of N. megapotamica, and the stereochemistry of α-bisabolol was determined. Two different chemotypes, based on volatile compounds, were identified, distinguished by the presence of isospathulenol, α-bisabolol, β-bisabolene, and (E)-nerolidol for chemotype A, and bicyclogermacrene and elemicin for chemotype B. A stereochemical analysis of chemotype A extract revealed (+)-α-bisabolol enantiomer. Histochemical tests of chemotypes showed similar results and suggested the presence of essential oil in idioblasts stained with the dye NADI. The analyses of chemotype A leaves by GC-MS revealed similar compositions for abaxial and adaxial surfaces, such pattern was also observed for chemotype B. Medium and high polarity metabolites showed high chemical similarities between the chemotypes, highlighting the presence of proanthocyanidins and glycosylated flavonoids (O- and C-glycosides). Thus, N. megapotamica produced distinct volatile chemotypes with highly conserved medium to high polarity compounds. Such results suggest that phenolic derivatives have a basal physiological function, while genetic or environmental differences lead to differentiation in volatile profiles of N. megapotamica.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201996PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078319PMC
February 2019

Engineering de novo anthocyanin production in Saccharomyces cerevisiae.

Microb Cell Fact 2018 Jul 3;17(1):103. Epub 2018 Jul 3.

Wageningen Plant Research, Wageningen University & Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.

Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/g, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.

Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.
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http://dx.doi.org/10.1186/s12934-018-0951-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029064PMC
July 2018

Engineering storage capacity for volatile sesquiterpenes in Nicotiana benthamiana leaves.

Plant Biotechnol J 2018 12 28;16(12):1997-2006. Epub 2018 May 28.

Wageningen Univ & Res, Wageningen Plant Res, Biosci, Wageningen, The Netherlands.

Plants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often carried out in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In this study, we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin-coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil seed-producing species castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co-expression of α-bisabolol synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from castor bean promoted storage of α-bisabolol in N. benthamiana mesophyll tissue more than 17-fold. A clear correlation was found between neutral lipids and storage of sesquiterpenes, using synthases for α-bisabolol, (E)-β-caryophyllene and α-barbatene. The co-localization of neutral lipids and α-bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants.
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http://dx.doi.org/10.1111/pbi.12933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230952PMC
December 2018

α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

J Biol Chem 2018 06 19;293(26):10042-10058. Epub 2018 Apr 19.

From the Departments of Medical Biochemistry,

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in leaf extracts, enabling the identification and biochemical characterization of an α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking -glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate ( = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.
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http://dx.doi.org/10.1074/jbc.RA118.001774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028973PMC
June 2018

Effect of dietary fiber (inulin) addition on phenolics and in vitro bioaccessibility of tomato sauce.

Food Res Int 2018 04 18;106:129-135. Epub 2017 Dec 18.

Faculty of Chemical and Metallurgical Engineering, Food Engineering Department, Istanbul Technical University, Maslak, 34469 Istanbul, Turkey. Electronic address:

The effect of the addition of inulin (5 and 10%) on the phenolic content and in vitro gastrointestinal digestion of tomato sauces has been investigated. Results have shown that the addition of inulin to tomato sauce significantly decreased the total phenolic content (57-68%), total flavonoid content (48-60%), and total antioxidant capacity (49-61%). Similarly, all assays of the sauce containing both 5% and 10% inulin, showed a slight decrease during in vitro gastrointestinal digestion of tomato sauces. Higher levels of inulin added to tomato sauce resulted in the greatest decrease in phenolic content, probably because of the interaction between inulin and phenolic compounds. To address the effects of inulin on the global metabolite profile of tomato sauce, an untargeted metabolomics approach was followed. Changes related to the presence of inulin suggest that inulin quenches a subset of unidentified compounds which are present in sauce but not in fruit, suggesting that inulin can contribute to the conservation of fruit properties in tomato sauce.
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http://dx.doi.org/10.1016/j.foodres.2017.12.050DOI Listing
April 2018

Methyl Perillate as a Highly Functionalized Natural Starting Material for Terephthalic Acid.

ChemistryOpen 2018 02 8;7(2):201-203. Epub 2018 Feb 8.

Wageningen Plant Research PO box 16 6700AA Wageningen The Netherlands.

Renewable commodity chemicals can be generated from plant materials. Often abundant materials such as sugars are used for this purpose. However, these lack appropriate functionalities and, therefore, they require extensive chemical modifications before they can be used as commodity chemicals. The plant kingdom is capable of producing an almost endless variety of compounds, including compounds with highly appropriate functionalities, but these are often not available in high quantities. It has been demonstrated that it is possible to produce functionalized plant compounds on a large scale by fermentation in microorganisms. This opens up the potential to exploit plant compounds that are less abundant, but functionally resemble commodity chemicals more closely. To elaborate this concept, we demonstrate the suitability of a highly functionalized plant compound, methyl perillate, as a precursor for the commodity chemical terephthalic acid.
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http://dx.doi.org/10.1002/open.201700178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803525PMC
February 2018

The effect of isabelin, a sesquiterpene lactone from Ambrosia artemisiifolia on soil microorganisms and human pathogens.

FEMS Microbiol Lett 2018 02;365(4)

Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, PO BOX 50, 6700 AB Wageningen, The Netherlands.

Ambrosia artemisiifolia L. (common ragweed) is an invasive weed, which is well known for the strong allergenic effect of its pollen as well as for its invasiveness and impact in crop fields (e.g. causing yield losses). This species produces a broad range of sesquiterpenoids. In recent years, new bioactive molecules have been discovered in this plant, e.g. isabelin, a sesquiterpene dilactone. The bioactivity of isabelin has been already demonstrated on allergy-related receptors and its inhibitory effect on seeds of various plant species. Isabelin was tested for potential antimicrobial effects by using a selection of soil-borne bacteria and fungi and three human pathogens as model organisms. For the majority of microorganisms tested, no antimicrobial activity of isabelin was observed. However, isabelin revealed strong antimicrobial activity against the Gram-positive soil bacterium Paenibacillus sp. and against the Gram-positive, multidrug-resistant Staphylococcus aureus. The observed inhibitory activity of isabelin can enlighten the importance to study similar compounds for their effect on human pathogens and on soil and rhizosphere microorganisms.
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http://dx.doi.org/10.1093/femsle/fny001DOI Listing
February 2018

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plant Physiol 2018 02 30;176(2):1862-1878. Epub 2017 Nov 30.

Wageningen Plant Research, Bioscience, 6700 AA, Wageningen, The Netherlands

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato () for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.
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http://dx.doi.org/10.1104/pp.17.01662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813534PMC
February 2018

Identification of major loci and genomic regions controlling acid and volatile content in tomato fruit: implications for flavor improvement.

New Phytol 2017 Jul 6;215(2):624-641. Epub 2017 Jun 6.

INRA, UR1052, GAFL, 67 Allée des Chênes Domaine Saint Maurice - CS60094, Montfavet Cedex, 84143, France.

Plant metabolites are important to world food security due to their roles in crop yield and nutritional quality. Here we report the metabolic profile of 300 tomato accessions (Solanum lycopersicum and related wild species) by quantifying 60 primary and secondary metabolites, including volatile organic compounds, over a period of 2 yr. Metabolite content and genetic inheritance of metabolites varied broadly, both within and between different genetic groups. Using genotype information gained from 10 000 single nucleotide polymorphism markers, we performed a metabolite genome-wide association mapping (GWAS) study. We identified 79 associations influencing 13 primary and 19 secondary metabolites with large effects at high resolution. Four genome regions were detected, highlighting clusters of associations controlling the variation of several metabolites. Local linkage disequilibrium analysis and allele mining identified possible candidate genes which may modulate the content of metabolites that are of significant importance for human diet and fruit consumption. We precisely characterized two associations involved in fruit acidity and phenylpropanoid volatile production. Taken together, this study reveals complex and distinct metabolite regulation in tomato subspecies and demonstrates that GWAS is a powerful tool for gene-metabolite annotation and identification, pathways elucidation, and further crop improvement.
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http://dx.doi.org/10.1111/nph.14615DOI Listing
July 2017

Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.

Sci Rep 2017 04 13;7(1):862. Epub 2017 Apr 13.

Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology, 6700 AB, Wageningen, The Netherlands.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.
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http://dx.doi.org/10.1038/s41598-017-00893-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429845PMC
April 2017

Processing black mulberry into jam: effects on antioxidant potential and in vitro bioaccessibility.

J Sci Food Agric 2017 Aug 8;97(10):3106-3113. Epub 2017 Feb 8.

Faculty of Chemical and Metallurgical Engineering, Food Engineering Department, Istanbul Technical University, Maslak, Istanbul, Turkey.

Background: Black mulberries (Morus nigra) were processed into jam on an industrialised scale, including the major steps of: selection of frozen black mulberries, adding glucose-fructose syrup and water, cooking, adding citric acid and apple pectin, removing seeds, and pasteurisation. Qualitative and quantitative determinations of antioxidants in black mulberry samples were performed using spectrophotometric methods, as well as HPLC- and LC-QTOF-MS-based measurements. These analyses included the determination of total polyphenolic content, % polymeric colour, total and individual anthocyanin contents, antioxidant capacity, and in vitro bioaccessibility in processing samples.

Results: Jam processing led to a significant reduction in total phenolics (88%), total flavonoids (89%), anthocyanins (97%), and antioxidant capacity (88-93%) (P < 0.05). Individual anthocyanin contents, determined using HPLC analysis, also showed a significant decrease (∼99% loss). In contrast, % recovery of bioaccessible total phenolics, anthocyanins, and antioxidant capacity (ABTS assay) increased after jam processing (16%, 12%, and 37%, respectively).

Conclusion: Fruit processing resulted in losses of polyphenols, anthocyanins, and antioxidant capacity of black mulberry jam. Optimisation of food processing could help to protect the phenolic compounds in fruits which might be helpful for the food industry to minimise the antioxidant loss and improve the final product quality. © 2016 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.8152DOI Listing
August 2017