Dynamic Metabolic Footprinting Reveals the Key Components of Metabolic Network in Yeast Saccharomyces cerevisiae

Metabolic footprinting offers a relatively easy approach to exploit the potentials of metabolomics for phenotypic characterization of microbial cells. To capture the highly dynamic nature of metabolites, we propose the use of dynamic metabolic footprinting instead of the traditional method which relies on analysis at a single time point. Using direct infusion-mass spectrometry (DI-MS), we could observe the dynamic metabolic footprinting in yeast S. cerevisiae BY4709 (wild type) cultured on 3 different C-sources (glucose, glycerol, and ethanol) and sampled along 10 time points with 5 biological replicates. In order to analyze the dynamic mass spectrometry data, we developed the novel analysis methods that allow us to perform correlation analysis to identify metabolites that significantly correlate over time during growth on the different carbon sources. Both positive and negative electrospray ionization (ESI) modes were performed to obtain the complete information about the metabolite content. Using sparse principal component analysis (Sparse PCA), we further identified those pairs of metabolites that significantly contribute to the separation. From the list of significant metabolite pairs, we reconstructed an interaction map that provides information of how different metabolic pathways have correlated patterns during growth on the different carbon sources

Mapping the interaction of Snf1 with TORC1 in Saccharomyces cerevisiae

Nutrient sensing and coordination of metabolic pathways are crucial functions for living cells. A combined analysis of the yeast transcriptome, phosphoproteome and metabolome is used to investigate the interactions between the Snf1 and TORC1 pathways under nutrient-limited conditions

Molecular characterisation of the GdhA- derivative of Pasteurella multocida B:2

Pasteurella multocida B:2 is an important veterinary pathogen causing fatal and acute haemorrhagic septicaemia (HS) in bovine. A live vaccine candidate, P. multocida B:2 GDH7 was reported to enable protection in cattle and buffaloes via intranasal (i. n.) administration. This potential vaccine was also reported to be self-transmitted from the vaccinated animal to the free-ranging animals allowing wider vaccination coverage. Prior to commercialisation, this potential vaccine requires further characterisation in accordance with the authoritative guidelines from the World Organisation for Animal Health (OIE). Hence, in this study, the potential vaccine strain, P. multocida B:2 GDH7 and the virulent parent strain were characterised through genomic and proteomic profiling. A crucial first step was to develop a sensitive yet simple and robust identification test to differentiate both strains which has been achieved by the development of a precise yet straightforward PCR method. In genomic profiling, Repetitive Extragenic Palindromic sequence-PCR (REP-PCR) was manipulated and both strains have a different display of genomic DNA band patterns. Some of the major OMPs were observed and prominent immunogens of P. multocida, OmpA and OmpH were observed to be expressed differently between these strains through SDS-PAGE analysis. In conclusion, a reproducible PCR detection method has enabled differentiation of both strains. Further characterisation of these strains shows a significantly different profile through genomic and proteomic profiling

Systems Biology of Yeast Lipid Metabolism

Lipid metabolism plays an important role in the development of many different life-style related diseases, such as type 2 diabetes and atherosclerosis, and understanding the molecular mechanisms behind regulation of lipid biosynthesis and degradation may lead to development of new therapies. In this project we undertook a global study of lipid metabolism in the eukaryotic model organism Saccharomyces cerevisiae. The objective of this project is to quantify how the fluxes in lipid metabolism of eukaryotic cells are controlled by different component of the regulatory network. Using systems biology approaches there was established a global regulatory model for lipid metabolism, and it was quantified how the fluxes toward different lipid components are regulated. Using different mutants that carry deletion in genes encoding key transcriptional factors and protein kinases involved in lipid regulation, the fluxes towards the different lipid components was perturbed. The wild-type yeast strain CEN.PK113-7D and the yeast mutants opi1∆, snf1∆, tor1∆, ino2∆, ino4∆, and ino2∆ino4∆ were grown in chemostat cultures at carbon or nitrogen-limited conditions and also high or low inositol-choline (IC) condition at a dilution rate of 0.1 h-1. At steady state conditions samples were withdrawn for analysis of the transcriptome, the metabolome and the lipidome. There was also developed 3 high-throughput methods for lipid quantification, i) for storage lipid monitoring at single-cell level using CARS microscopy, ii) for lipid classes analysis based on microwave-assisted extraction, HPLC-CAD, and iii) for fatty acids species analysis based on microwave-assisted derivatization. Through combined measurements of the transcriptome, the metabolome, the lipidome and the fluxome it was possible to obtain a large dataset that could be used to identify correlations between the different components such as the co-influences of Snf1-IC effects, INO-level, and Snf1-TORC1 effects on yeast lipid metabolism

Transcriptome analysis reveals candidate genes involved in luciferin metabolism in Luciola aquatilis (Coleoptera: Lampyridae)

Bioluminescence, which living organisms such as fireflies emit light, has been studied extensively for over half a century. This intriguing reaction, having its origins in nature where glowing insects can signal things such as attraction or defense, is now widely used in biotechnology with applications of bioluminescence and chemiluminescence. Luciferase, a key enzyme in this reaction, has been well characterized; however, the enzymes involved in the biosynthetic pathway of its substrate, luciferin, remains unsolved at present. To elucidate the luciferin metabolism, we performed a de novo transcriptome analysis using larvae of the firefly species, Luciola aquatilis. Here, a comparative analysis is performed with the model coleopteran insect Tribolium casteneum to elucidate the metabolic pathways in L. aquatilis. Based on a template luciferin biosynthetic pathway, combined with a range of protein and pathway databases, and various prediction tools for functional annotation, the candidate genes, enzymes, and biochemical reactions involved in luciferin metabolism are proposed for L. aquatilis. The candidate gene expression is validated in the adult L. aquatilis using reverse transcription PCR (RT-PCR). This study provides useful information on the bio-production of luciferin in the firefly and will benefit to future applications of the valuable firefly bioluminescence system

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP–FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications

Amplification and bioinformatics analysis of conserved FAD-binding region of L-amino acid oxidase (LAAO) genes in gastropods compared to other organisms

This study aimed to investigate the conserved FAD-binding region of the L-amino acid oxidase (LAAO) genes in twelve gastropod genera commonly found in Thailand compared to those in other organisms using molecular cloning, nucleotide sequencing and bioinformatics analysis. Genomic DNA of gastropods and other invertebrates was extracted and screened using primers specific to the conserved FAD-binding region of LAAO. The amplified 143-bp fragments were cloned and sequenced. The obtained nucleotide sequences of 21 samples were aligned and phylogenetically compared to the LAAO-conserved FAD-binding regions of 210 other organisms from the NCBI database. Translated amino acid sequences of these samples were used in phylogenetics and pattern analyses. The phylogenetic trees showed clear separation of the conserved regions in fungi, invertebrates, and vertebrates. Alignment of the conserved 47-amino-acid FAD-binding region of the LAAOs showed 150 unique sequences among the 231 samples and these patterns were different from those of other flavoproteins in the amine oxidase family. An amino acid pattern analysis of five sub-regions (bFAD, FAD, FAD-GG, GG, and aGG) within the FAD-binding sequence showed high variation at the FAD-GG sub-region. Pattern analysis of secondary structures indicated the aGG sub-region as having the highest structural variation. Cluster analysis of these patterns revealed two major clusters representing the mollusc clade and the vertebrate clade. Thus, molecular phylogenetics and pattern analyses of sequence and structural variations could reflect evolutionary relatedness and possible structural conservation to maintain specific function within the FAD-binding region of the LAAOs in gastropods compared to other organisms. Keywords: L-amino acid oxidase (LAAO), FAD-binding region, Gastropods, Pattern analysis, Clusterin

Understanding Snail Mucus Biosynthesis and Shell Biomineralisation through Genomic Data Mining of the Reconstructed Carbohydrate and Glycan Metabolic Pathways of the Giant African Snail (<i>Achatina fulica</i>)

The giant African snail (Order Stylommatophora: Family Achatinidae), Achatina fulica (Bowdich, 1822), is the most significant and invasive land snail pest. The ecological adaptability of this snail involves high growth rate, reproductive capacity, and shell and mucus production, driven by several biochemical processes and metabolism. The available genomic information for A. fulica provides excellent opportunities to hinder the underlying processes of adaptation, mainly carbohydrate and glycan metabolic pathways toward the shell and mucus formation. The authors analysed the 1.78 Gb draft genomic contigs of A. fulica to identify enzyme-coding genes and reconstruct biochemical pathways related to the carbohydrate and glycan metabolism using a designed bioinformatic workflow. Three hundred and seventy-seven enzymes involved in the carbohydrate and glycan metabolic pathways were identified based on the KEGG pathway reference in combination with protein sequence comparison, structural analysis, and manual curation. Fourteen complete pathways of carbohydrate metabolism and seven complete pathways of glycan metabolism supported the nutrient acquisition and production of the mucus proteoglycans. Increased copy numbers of amylases, cellulases, and chitinases highlighted the snail advantage in food consumption and fast growth rate. The ascorbate biosynthesis pathway identified from the carbohydrate metabolic pathways of A. fulica was involved in the shell biomineralisation process in association with the collagen protein network, carbonic anhydrases, tyrosinases, and several ion transporters. Thus, our bioinformatic workflow was able to reconstruct carbohydrate metabolism, mucus biosynthesis, and shell biomineralisation pathways from the A. fulica genome and transcriptome data. These findings could reveal several evolutionary advantages of the A. fulica snail, and will benefit the discovery of valuable enzymes for industrial and medical applications