Paul D Fraser

Royal Holloway, University of London, Эгхем, England, United Kingdom

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Publications (110)476.12 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus indicus and Bacillus firmus synthesize C30 carotenoids via farnesyl pyrophosphate (FPP) forming apophytoene the first committed step in the pathway. The end products of the pathways are derivedvia diapolycopen-monoic acid or diapolycopen-dioic acid, respectively, as intermediates. The genomes of both bacteria have been sequenced and the genes for their early terpenoid and specific carotenoid pathways annotated. All genes for a functional 1-deoxy-D-xylulose 5-phosphate synthase pathway were identified in both species whereas the genes of the mevalonate pathway were absent. The gene for direct carotenoid synthesis and conversion were found on a gene cluster which are differently organised. The genes involved in the formation of the carotenoid cores have been assigned by functional complementation in Escherichia coli. This bacterium was co-transformed with a plasmid mediating the formation of the putative substrate and a second plasmid with the gene of interest. Carotenoid products in the transformants were determined by HPLC. By this approach, the genes for a diapophytoene synthase, crtM, for a diapophytoene desaturase, crtNa, for a diapolycopene ketolase, crtNb, and for a diapolycopene aldehyde oxidase, crtNc, were identified. The three crtN genes are closely related and belong to the crtI gene family with a similar reaction mechanism of their enzyme products. Additional genes for the modification of the carotenoid skeleton, diapolycopenoic acids encoding glycosyl transferases and acyl transferases were identified in comparison to the corresponding genes from other bacteria.
    Microbiology (Reading, England). 10/2014;
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    ABSTRACT: Application of mass spectrometry enables the detection of metabolic differences between groups of related organisms. Differences in the metabolic fingerprints of wild-type Solanum lycopersicum and three monogenic mutants, ripening inhibitor (rin), non-ripening (nor) and Colourless non-ripening (Cnr), of tomato are captured with regard to ripening behaviour. A high-resolution tandem mass spectrometry system coupled to liquid chromatography produced a time series of the ripening behaviour at discrete intervals with a focus on changes post-anthesis. Internal standards and quality controls were used to ensure system stability. The raw data of the samples and reference compounds including study protocols have been deposited in the open metabolomics database MetaboLights via the metadata annotation tool Isatab to enable efficient re-use of the datasets, such as in metabolomics cross-study comparisons or data fusion exercises.
    Scientific Data. 09/2014; Scientific Data 1, Article number: 140029(1):140029.
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    ABSTRACT: Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered.
    Food Chemistry 04/2014; 149:121-8. · 3.33 Impact Factor
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    ABSTRACT: Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large-scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant-derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.
    Biotechnology Journal 02/2014; · 3.71 Impact Factor
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    ABSTRACT: The rapid advances in sequencing technologies over the last decade have enabled routine sequencing of microbial genomes. Despite notable achievements, metabolomics/metabolite profiling has not progressed with the same rapidity, which in part is due to the intrinsic complex chemical nature of the metabolome. However, well characterised metabolomes are essential if a comprehensive understanding of biological function and biotechnological applications are to be revealed and implemented. In the present study a hyphenated MS metabolite profiling procedure has been developed, predominantly for Bacillus species. The approach has been systematic in its development, delivering optimised procedures for the quenching of bacterial metabolism, extraction of metabolites, the separation and detection of components as well as data analysis, integration and visualisation workflows. Collectively, the procedure has enabled the detection of 27 % of the predicted Bacillus subtilis metabolome in the industrial HU36 strain. The analytical platform developed has been used to assess the chemotype of commercially used probiotic Bacillus strains, including a novel pigmented Bacillus strain HU36 that has potential either as a probiotic or source of antioxidants. The results are discussed in a biochemical context, revealing: (i), specific metabolic networks associated with pigment biosynthesis in HU36 and (ii), biotechnological applications through the demonstration of substantial equivalence.
    Metabolomics 02/2014; 10(1). · 4.43 Impact Factor
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    ABSTRACT: The processing of dry-cured ham results in an intense proteolysis that leads to the generation of peptides of different sizes and composition, which have been shown to be biologically active. In this study, a total of ninety-three peptides mainly derived from actin, β-enolase, myosin heavy chain, and creatine kinase proteins have been identified from a size-exclusion chromatography (SEC) fraction that resulted as antioxidant by using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) and nESI-ion trap mass spectrometry. Several of the identified peptides have been synthesised and their antioxidant activity tested in vitro by using DPPH radical-scavenging assay and reducing power analysis. The peptide with sequence SNAAC showed the best results with an IC50 of 75.2 μM in DPPH radical-scavenging assay and 205 μM in ferric-reducing antioxidant power analysis, very good when comparing with the positive control 2,6-di-tert-butyl-4-methyl phenol (BHT) that showed an IC50 of 358.5 μM and 90.3 μM, respectively, in the different assays. These results suggest that Spanish dry-cured ham represents an important source of powerful antioxidant peptides which due to their natural characteristics may represent a highly valuable alternative in human health.
    Food Research International 02/2014; 56:68–76. · 3.05 Impact Factor
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    ABSTRACT: The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.
    Journal of Visualized Experiments 01/2014;
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    ABSTRACT: In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as M. tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for M. smegmatis, M. phlei and M. bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.
    Journal of Microbiological Methods. 01/2014;
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    ABSTRACT: Tomato and its processed products are one of the most widely consumed fruits. Its domestication, however, has resulted in the loss of some 95% of the genetic and chemical diversity of wild relatives. In order to elucidate this diversity, exploit its potential for plant breeding, as well as understand its biological significance, analytical approaches have been developed, alongside the production of genetic crosses of wild relatives with commercial varieties. In this article, we describe a multi-platform metabolomic analysis, using NMR, mass spectrometry and HPLC, of introgression lines of Solanum pennellii with a domesticated line in order to analyse and quantify alleles (QTL) responsible for metabolic traits. We have identified QTL for health-related antioxidant carotenoids and tocopherols, as well as molecular signatures for some 2000 compounds. Correlation analyses have revealed intricate interactions in isoprenoid formation in the plastid that can be extrapolated to other crop plants.
    Scientific Reports 01/2014; 4:3859. · 5.08 Impact Factor
  • Ron Wehrens, Elisabete Carvalho, Paul D. Fraser
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    ABSTRACT: For those chemical compounds absorbing in the UV–Vis region and not readily applicable to routine mass spectrometry ionisation methods, liquid chromatography coupled to diode array detection is a convenient platform to perform metabolite profiling. Data processing by hand is labour-intensive and error prone. In the present study a strategy based on multivariate curve resolution, and its implementation in an R package called alsace are described. The final result of an analysis is a table containing peak heights or peak areas for all features of the individual injections. The capabilities of the software, providing elements such as splitting the data into separate, possibly overlapping time windows, merging the results of the individual time windows, and parametric time warping to align features, are illustrated using a cassava-derived data set.
    Metabolomics 01/2014; · 4.43 Impact Factor
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    ABSTRACT: Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.
    The Plant Cell 11/2013; · 9.25 Impact Factor
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    ABSTRACT: The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.
    Biotechnology Journal 10/2013; 8(10):1159-1171. · 3.71 Impact Factor
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    ABSTRACT: The composition of carotenoids, chlorophyll derivatives and tocopherols in raspberries of different varieties, including yellow and red varieties, over different ripening stages has been studied. The profile of pigments in ripening raspberries changes drastically, with a dramatic decrease of β-carotene and chlorophyll derivatives, the xanthophyll lutein has also decreased but not to the same extent. In contrast esterified lutein increased and is present in ripe raspberries esterified with saturated fatty acids with C8-C16 chains. Ripe raspberries contain considerable amounts of free lutein, esterified lutein, and tocopherols (up to 20, 49 and 366mg/kg dry weight, respectively). The different samples analysed show different contents of carotenoids and tocopherols. Whether the differences arise from the variety or other factors such as the environmental conditions needs to be ascertained but isoprenoids should not be neglected when considering raspberry antioxidant and nutraceutical composition.
    Food Chemistry 08/2013; 139(1-4):744-52. · 3.33 Impact Factor
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    ABSTRACT: Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.
    Protoplasma 06/2013; · 2.86 Impact Factor
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    ABSTRACT: The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5units of absorbance at 700nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.
    Food Chemistry 06/2013; 138(2-3):1282-8. · 3.33 Impact Factor
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    Leticia Mora, Peter M Bramley, Paul D Fraser
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    ABSTRACT: A key global challenge for plant biotechnology is addressing food security, whereby provision must be made to feed 9 billion people with nutritional feedstuffs by 2050. To achieve this step-change in Agricultural production new crop varieties are required that are tolerant to environmental stresses imposed by climate change, have better yields, are more nutritious and require less resource input. Genetic modification (GM) and Marker Assisted Screening (MAS) will need to be fully utilised to deliver these new crop varieties. To evaluate these varieties both in terms of environmental and food safety and the ration design of traits a systems level characterisation is necessary. To link the transcriptome to the metabolome, quantitative proteomics is required. Routine quantitative proteomics is an important challenge. Gel-based densitometry and MS analysis after stable isotope labeling have been employed. In the present article we describe the application of a label-free approach that can be used in combination with SDS-PAGE and reverse-phase chromatography to evaluate the changes in the proteome of new crop varieties. The workflow has been optimised for protein coverage, accuracy, and robustness, then its application demonstrated using a GM tomato variety engineered to deliver nutrient dense fruit. This article is protected by copyright. All rights reserved.
    Proteomics 04/2013; · 4.43 Impact Factor
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    ABSTRACT: Strigolactones (SLs) are a class of phytohormones controlling shoot branching. In potato (Solanum tuberosum), tubers develop from underground stolons, diageotropic stems which originate from basal stem nodes. As the degree of stolon branching influences the number and size distribution of tubers, it was considered timely to investigate the effects of SL production on potato development and tuber life cycle. Transgenic potato plants were generated in which the CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) gene, key in the SL biosynthetic pathway, was silenced by RNA interference (RNAi). The resulting CCD8-RNAi potato plants showed significantly more lateral and main branches than control plants, reduced stolon formation, together with a dwarfing phenotype and a lack of flowering in the most severely affected lines. New tubers were formed from sessile buds of the mother tubers. The apical buds of newly formed transgenic tubers grew out as shoots when exposed to light. In addition, we found that CCD8 transcript levels were rapidly downregulated in tuber buds by the application of sprout-inducing treatments. These results suggest that SLs could have an effect, solely or in combination with other phytohormones, in the morphology of potato plants and also in controlling stolon development and maintaining tuber dormancy.
    New Phytologist 03/2013; · 6.74 Impact Factor
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    ABSTRACT: The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long chain prenyl diphosphates. We report the cloning and characterisation of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. Solanesyl diphosphate synthase, SlSPS, is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene (SlDPS), encodes a long-chain prenyl diphosphate synthase with a different subcellular localisation from SlSPS and can utilise geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in E. coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to 9 and 10 isoprene units, respectively. In planta, constitutive over expression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whilst silencing SlDPS did not affect leaf appearance but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long chain prenyl diphosphate synthases in tomato.
    Biochemical Journal 11/2012; · 4.65 Impact Factor
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    ABSTRACT: Novel sequences exhibiting in vitro ACE inhibitory activity as well as in vivo antihypertensive activity were identified from Spanish dry-cured ham. Water soluble peptide extracts from dry-cured ham were purified by size-exclusion chromatography and reversed-phase high performance liquid chromatography and then, further identification of sequences was carried out by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 73 peptide sequences were identified from active fractions presenting 100% homology with different Sus scrofa skeletal muscle proteins. All identified peptides showed Mr between 374 and 1610 and amino acid sequences between 5 and 14 amino acids in length. Considering the low molecular mass and structural requirements for ACE inhibition some of the identified peptides were synthesised and their IC(50) calculated. The most potent peptide was found to be AAATP (IC(50) value of 100μM). This peptide also showed good in vivo activity because it decreased systolic blood pressure by -25.62±4.5mmHg (p<0.05) in spontaneous hypertensive rats after 8h administration. Other sequences yielded a moderate ACE inhibition. Results from this study show that Spanish dry-cured ham may represent a source of natural peptides with potential benefit for human health.
    Journal of proteomics 10/2012; · 5.07 Impact Factor

Publication Stats

3k Citations
476.12 Total Impact Points

Institutions

  • 1996–2014
    • Royal Holloway, University of London
      • • Department of Biological Sciences
      • • Division of Biochemistry
      Эгхем, England, United Kingdom
  • 1994–2014
    • University of London
      • School of Biological Sciences
      Londinium, England, United Kingdom
  • 2013
    • James Hutton Institute
      • Cell and Molecular Science
      Aberdeen, Scotland, United Kingdom
  • 1998–2012
    • Goethe-Universität Frankfurt am Main
      • Institut für Molekulare Biowissenschaften
      Frankfurt am Main, Hesse, Germany
    • Tokyo Institute of Technology
      Edo, Tōkyō, Japan
  • 2006–2011
    • Scottish Crop Research Institute
      Aberdeen, Scotland, United Kingdom
    • Zhejiang University
      Hang-hsien, Zhejiang Sheng, China
  • 2009
    • Japan Women's University
      Edo, Tōkyō, Japan
  • 1995–2005
    • University of Bristol
      • School of Biological Sciences
      Bristol, ENG, United Kingdom
  • 1992–1993
    • Universität Konstanz
      • Plant Physiology and Plant Biochemistry
      Konstanz, Baden-Wuerttemberg, Germany