Establishing a Comprehensive Toolbox for Isotopic Labelling Studies on Terpene Synthases

The cumulative doctoral thesis "Establishing a Comprehensive Toolbox for Isotopic Labelling Studies on Terpene Synthases" describes the synthesis and application of isotopically labelled compounds for the systematic in vitro investigation of recombinant terpene synthases to target both cyclisation mechanism and product structure. Methodically, the known approach of enantioselectively deuterated oligoprenyl diphosphate substrates was further developed by the addition of 13C-labelling, which led to a more sensitive detection of the labelled product by NMR. With a stereochemical anchor of known absolute configuration installed in the substrate and untouched by the enzymatic cyclisation mechanism, it is possible to infer the absolute configuration of the terpene product by following the incorporation of deuterium into the diastereotopic hydrogen positions. By combining chemical and enzymatic synthesis, it was finally possible to label every methylene group of the common terpene precursors by 13C and 2H in an enantioselective fashion. These extensions improve both feasibility and robustness of this method, which contributes to the challenging structure elucidation of terpene natural products, including their difficult to address absolute configurations. Depending on the cyclisation mechanism, also the stereochemical course of hydrogen movements can be delineated. Connected to the expanding labelling possibilities, several newly identified terpene synthases from bacteria and fungi have been addressed covering various aspects of their catalysis such as substrate or product specificity, repetitive mechanistic motifs and stereochemical issues. The structural variety of the known and newly identified natural products thereby inspired further studies like tailored labelling experiments, site-directed mutagenesis, chemical modifications and the investigation of EI-MS fragmentation mechanisms. With few publications dealing with other aspects of natural product chemistry such as fungal aromatic volatiles, lignin degradation and selected aspects of the secondary metabolism of marine Roseobacter group bacteria also being included in this work, the main focus lays on a deepened understanding of terpene synthase reactions. The isotopically labelled substrates introduced in this study thereby represent a valuable experimental tool towards a comprehensive picture of these astonishing enzymes that create the largest group of natural products

Natural Medicine in Therapy

Enter For a long time, natural medicine has been used as a therapeutic therapy based on generations of indigenous practices. Today the rise in natural remedies has been largely driven by public demand and billions of dollars are spent annually on herbal medicines. It is therefore important to document the effectiveness of natural medicine, its potential side effects, and potential interactions

Metabolomic profiling of antibiofilm compounds from fungal endophytes derived from Scottish seaweeds

In the search for a new antimicrobial bioactive compound from natural source against both biofilm-forming Staphylococcus aureus and Pseudomonas aeruginosa, endophytic fungi associated with seaweeds are an interesting source of bioactive secondary metabolites. Twenty-four endophytes were isolated from eight different Scottish seaweeds namely; Ascophyllum nodosum, Laminaria hyperborean, Ulva intestinalis, Ulva lactuca, Himanthalia elongata, Fucus vesiculosis, Fucus serratus, and Fucus spiralis. Endophytes responsible for producing bioactive compounds were identified using internal transcribed spacer (ITS) gene sequencing. Depending on their biological activity and chemical profiles, three endophytic fungi, namely; Dendryphiella salina (D. salina), Hypoxylon rubiginosum (H. rubiginosum), and Mariannaea elegans (M. elegance) were identified and subjected for media optimisation studies on five liquid media and four solid media in three different incubation periods. For liquid media; this included: malt extract broth with and without sea salt, Wickersham liquid media with and without sea salt and lastly, marine broth. While for the solid media, rice and oat, both with and without sea salt were used. The chemical profile for each extract was monitored using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). Antimicrobial activity was monitored using both AlamarBlue® and planktonic assays. The appropriate medium and the incubation period were chosen for a scale-up of the selected endophytic fungus according to the following factors: 1) higher yield, 2) improved antimicrobial activity, and 3) diverse chemical profile. Based on these factors, D. salina was chosen for scale-up and isolation work. D. salina was grown on malt extract with salt and oat without salt. The scaled-up extracts of D. salina were subjected to liquid-liquid partitioning followed by fractionation using either medium pressure flash chromatography or flash chromatographic technique. The first fractionation of the scaled-up extracts was tested against both biofilm-forming S. aureus and P. aeruginosa and analysed using 1H NMR and LC-HRMS. The HRMS data was processed using MZmine followed by dereplication using an in-house method then subjected to orthogonal partial least square-discriminant analysis (OPLS-DA). OPLS-DA results were used determine the antimicrobial active secondary metabolites. As a result, eight compounds were isolated from the bioactive fractions of D. salina. D. salina grown on malt extract broth yielded linoleic acid, orsellinic acid, orcinol and 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid. While D. salina inoculated on oat solid media afforded the acetonide and glycerylglycoside derivatives of linoleic acid along with the peptide turnagainolide A. All isolated compounds, except for linoleic acid and its glycerol congener, displayed antibiofilm activity with MBEC (Minimum Biofilm Eradication Concentration) values between 45 and 360 µM concentrations against biofilm-forming bacteria S. aureus and P. aeruginosa. F10-2 (turnagainolide A) and F10-8 (gingerglycolipid B stereoisomer) displayed the highest potency with MIC and MBEC values between 45 and 75 µM concentrations. Conclusion: Endophytic D. salina derived from Scottish seaweed L. hyperborea grown on malt extract broth and oat solid media showed the ability to produce compounds with antimicrobial bioactivity against biofilm-forming bacteria. In addition, three new antimicrobial natural products: 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid along with an acetonide and glycerylglycoside derivatives of linoleic acid were isolated from D. salina extracts. Thus, marine endophytic fungi are promising source of bioactive compounds.In the search for a new antimicrobial bioactive compound from natural source against both biofilm-forming Staphylococcus aureus and Pseudomonas aeruginosa, endophytic fungi associated with seaweeds are an interesting source of bioactive secondary metabolites. Twenty-four endophytes were isolated from eight different Scottish seaweeds namely; Ascophyllum nodosum, Laminaria hyperborean, Ulva intestinalis, Ulva lactuca, Himanthalia elongata, Fucus vesiculosis, Fucus serratus, and Fucus spiralis. Endophytes responsible for producing bioactive compounds were identified using internal transcribed spacer (ITS) gene sequencing. Depending on their biological activity and chemical profiles, three endophytic fungi, namely; Dendryphiella salina (D. salina), Hypoxylon rubiginosum (H. rubiginosum), and Mariannaea elegans (M. elegance) were identified and subjected for media optimisation studies on five liquid media and four solid media in three different incubation periods. For liquid media; this included: malt extract broth with and without sea salt, Wickersham liquid media with and without sea salt and lastly, marine broth. While for the solid media, rice and oat, both with and without sea salt were used. The chemical profile for each extract was monitored using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). Antimicrobial activity was monitored using both AlamarBlue® and planktonic assays. The appropriate medium and the incubation period were chosen for a scale-up of the selected endophytic fungus according to the following factors: 1) higher yield, 2) improved antimicrobial activity, and 3) diverse chemical profile. Based on these factors, D. salina was chosen for scale-up and isolation work. D. salina was grown on malt extract with salt and oat without salt. The scaled-up extracts of D. salina were subjected to liquid-liquid partitioning followed by fractionation using either medium pressure flash chromatography or flash chromatographic technique. The first fractionation of the scaled-up extracts was tested against both biofilm-forming S. aureus and P. aeruginosa and analysed using 1H NMR and LC-HRMS. The HRMS data was processed using MZmine followed by dereplication using an in-house method then subjected to orthogonal partial least square-discriminant analysis (OPLS-DA). OPLS-DA results were used determine the antimicrobial active secondary metabolites. As a result, eight compounds were isolated from the bioactive fractions of D. salina. D. salina grown on malt extract broth yielded linoleic acid, orsellinic acid, orcinol and 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid. While D. salina inoculated on oat solid media afforded the acetonide and glycerylglycoside derivatives of linoleic acid along with the peptide turnagainolide A. All isolated compounds, except for linoleic acid and its glycerol congener, displayed antibiofilm activity with MBEC (Minimum Biofilm Eradication Concentration) values between 45 and 360 µM concentrations against biofilm-forming bacteria S. aureus and P. aeruginosa. F10-2 (turnagainolide A) and F10-8 (gingerglycolipid B stereoisomer) displayed the highest potency with MIC and MBEC values between 45 and 75 µM concentrations. Conclusion: Endophytic D. salina derived from Scottish seaweed L. hyperborea grown on malt extract broth and oat solid media showed the ability to produce compounds with antimicrobial bioactivity against biofilm-forming bacteria. In addition, three new antimicrobial natural products: 2,5-dihydroxy-3-(hydroxymethyl)benzoic acid along with an acetonide and glycerylglycoside derivatives of linoleic acid were isolated from D. salina extracts. Thus, marine endophytic fungi are promising source of bioactive compounds

Phytochemical Omics in Medicinal Plants

Medicinal plants are used to treat diseases and provide health benefits, and their applications are increasing around the world. A huge array of phytochemicals have been identified from medicinal plants, belonging to carotenoids, flavonoids, lignans, and phenolic acids, and so on, with a wide range of biological activities. In order to explore our knowledge of phytochemicals with the assistance of modern molecular tools and high-throughput technologies, this book collects recent innovative original research and review articles on subtopics of mechanistic insights into bioactivities, treatment of diseases, profiling, extraction and identification, and biotechnology