[Show abstract][Hide abstract] ABSTRACT: Proteolysis is a complex and dynamic process which takes place throughout the whole dry-cured processing due to the action of endogenous muscle peptidases, and results in the generation of a high number of small peptides and free amino acids responsible for the final quality of dry-cured ham. In this study, a total of sixty-eight peptides derived from the ubiquitin-60S ribosomal protein have been identified in dry-cured ham at 2, 3.5, 5, 6.5, and 9 months of processing using various chromatographic separations and a quadrupole/time-of-flight mass spectrometer in tandem. Some of the identified peptides have been detected during the whole process, whereas a total of fourteen of them were exclusively identified at 9 months of curing. The presence of any of these peptides could be a good indicative that dry-cured ham pieces have reached a minimum curing process of 9 months. The study of the generated peptides has contributed both to a better knowledge of proteolysis evolution and the endogenous enzymes participating, and to determine their potential to be used as quality markers to monitor the processing time.
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2014 · Journal of Microbiological Methods
[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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Full-text · Article · May 2014 · Journal of Visualized Experiments
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: The work described in this paper has used bacterial transgenes (crtI and Hmgr-CoA) which have the potential to increase oil quality in sunflower (Helianthus annuus L.) if an efficient transformation procedure was in place. Optimized procedures for the callus induction from hypocotyl and cotyledon explants, regeneration capacity of sunflower genotypes and transformation to intact embryogenic axis have been established in order to facilitate studies of transformation and production of genetic variability. Callus formation was induced easily from hypocotyl and cotyledon explants. Although cotyledon explants produced low amount of callus per explant, the somatic embryo and direct shoot regeneration capacity of cotyledons were generally much higher than that experienced with hypocotyl explants. Only root regeneration was obtained from hypocotyl explants. Regeneration of embryo and shoot varied from 0 - 29% depended on the genotype and explant. For transformation of sunflower, intact embryogenic axis were dissected from seeds and cocultivated with Agrobacterium tumefaciens. Transgenic sunflower lines expressing either the Erwinia uredovora phytoene desaturase (crtI) gene or hydroxymethylglutaryl-CoA (Hmgr-CoA) reductase genes have been obtained. Possible transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by PCR and nptII. Green shoots were transferred to rooting medium and showed early flowering in in vitro culture conditions. The transformation system is more efficient (90-100%) than previous reports and has shown the incorporation of effector transgenes.
[Show abstract][Hide abstract] 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.
Full-text · Article · Mar 2014 · Biotechnology Journal
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Feb 2014 · Food Research International
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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 ofSolanum 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.
Full-text · Article · Jan 2014 · Scientific Reports
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Dry-cured meat products experience an intense proteolysis phenomenon during their processing. Products like dry-cured ham, that experience processing times longer than 10 months, show an extensive breakdown of major proteins and the generation of a high number of small peptides and finally, large amounts of free amino acids. Proteomic techniques have been successfully applied to the identification of the generated peptides and their sequencing. These data are essential for a better understanding of proteolytic enzymes and their reaction during processing. This approach should reveal key biomarker peptides controlling the process and establish strategies to drive and optimise enzyme reactions for the production of optimal quality products.
No preview · Article · Nov 2013 · Food Research International