Development of an innovative molecular networking-based approach for the discovery and targeted isolation of new bioactive metabolites from higher plants

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.

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Feature-Based Molecular Networking to Target the Isolation of New Caffeic Acid Esters from Yacon (Smallanthus sonchifolius, Asteraceae)

Metabolites ◽

10.3390/metabo10100407 ◽

2020 ◽

Vol 10 (10) ◽

pp. 407

Author(s):

Guillermo F. Padilla-González ◽

Nicholas J. Sadgrove ◽

Gari V. Ccana-Ccapatinta ◽

Olga Leuner ◽

Eloy Fernandez-Cusimamani

Keyword(s):

Mass Spectrometry ◽

Caffeic Acid ◽

High Resolution Mass Spectrometry ◽

Chemical Diversity ◽

Bioactive Metabolites ◽

Molecular Networking ◽

Smallanthus Sonchifolius ◽

Feature Based ◽

Caffeic Acid Esters ◽

Acid Esters

Smallanthus sonchifolius (yacon) is an edible tuberous Andean shrub that has been included in the diet of indigenous people since before recorded history. The nutraceutical and medicinal properties of yacon are widely recognized, especially for the improvement of hyperglycemic disorders. However, the chemical diversity of the main bioactive series of caffeic acid esters has not been explored in detail. In this metabolomics study, we applied the latest tools to facilitate the targeted isolation of new caffeic acid esters. Using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we analyzed extracts from different organs (roots, vascular tissues of the stems, stem epidermis, leaves, bracts, and ray flowers) and followed a feature-based molecular networking approach to characterize the structural diversity of caffeic acid esters and recognize new compounds. The analysis identified three potentially new metabolites, one of them confirmed by isolation and full spectroscopic/spectrometric assignment using nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), and MS/MS. This metabolite (5-O-caffeoyl-2,7-anhydro-d-glycero-β-d-galacto-oct-2-ulopyranosonic acid), along with eight known caffeic acid esters, was isolated from the roots and stems. Furthermore, based on detailed tandem MS analyses, we suggest that the two isomeric monocaffeoyl-2,7-anhydro-2-octulopyranosonic acids found in yacon can be reliably distinguished based on their characteristic MS2 and MS3 spectra. The outcome of the current study confirms the utility of feature-based molecular networking as a tool for targeted isolation of previously undescribed metabolites and reveals the full diversity of potentially bioactive metabolites from S. sonchifolius.

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Bioactive Molecular Networking for Mapping the Antimicrobial Constituents of the Baltic Brown Alga Fucus vesiculosus

Marine Drugs ◽

10.3390/md18060311 ◽

2020 ◽

Vol 18 (6) ◽

pp. 311 ◽

Cited By ~ 1

Author(s):

Larissa Buedenbender ◽

Francesca Anna Astone ◽

Deniz Tasdemir

Keyword(s):

Antimicrobial Activity ◽

Brown Alga ◽

Cell Walls ◽

Algal Cell ◽

Fucus Vesiculosus ◽

Bioactive Metabolites ◽

Human Pathogens ◽

Molecular Networking ◽

Intertidal Zones ◽

The Baltic

The brown alga Fucus vesiculosus is common to the intertidal zones of the Baltic Sea, where it is exposed to high fouling pressures by microorganisms. Our previous studies showed, repeatedly, the consistent antimicrobial activity of F. vesiculosus crude extracts against human pathogens, while untargeted metabolomics analyses have revealed a variety of metabolites. In this study, we applied the UPLC-QToF-MS/MS-based “bioactive molecular networking” (BMN) concept on the most bioactive n-hexane and n-butanol subextracts of Baltic F. vesiculosus coupled with in silico dereplication tools to identify the compounds responsible for antimicrobial activity. The first antimicrobial cluster identified by BMN was galactolipids. Our targeted isolation efforts for this class led to the isolation of six monogalactosyldiacylglycerol (MGDG) derivatives (1–6) and one digalactosyldiacylglycerol (DGDG, 7). The MGDGs 5 and 6 and the DGDG 7 exhibited activity against Staphylococcus aureus. The second compound class with high bioactivity was phlorotannins. In particular, phlorethol-type phlorotannins showed high correlations with antimicrobial activity based on the BMN approach, and two phlorotannins (8–9) were isolated. This study shows that antimicrobial components of F. vesiculosus reside in the algal cell walls and membranes and that BMN provides a complementary tool for the targeted isolation of bioactive metabolites.

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Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea

Marine Drugs ◽

10.3390/md18070366 ◽

2020 ◽

Vol 18 (7) ◽

pp. 366

Author(s):

Reza Mohsenian Kouchaksaraee ◽

Mahdi Moridi Farimani ◽

Fengjie Li ◽

Melika Nazemi ◽

Deniz Tasdemir

Keyword(s):

Antibacterial Activity ◽

Nmr Spectroscopy ◽

1H Nmr ◽

Crude Extract ◽

Persian Gulf ◽

1H Nmr Spectroscopy ◽

Bioactive Metabolites ◽

Molecular Networking ◽

Isolation And Characterization ◽

The Persian Gulf

The geographic position, highly fluctuating sea temperatures and hypersalinity make Persian Gulf an extreme environment. Although this unique environment has high biodiversity dominated by invertebrates, its potential in marine biodiscovery has largely remained untapped. Herein, we aimed at a detailed analysis of the metabolome and bioactivity profiles of the marine sponge Axinella sinoxea collected from the northeast coast of the Persian Gulf in Iran. The crude extract and its Kupchan subextracts were tested in multiple in-house bioassays, and the crude extract and its CHCl3-soluble portion showed in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium (Efm). A molecular networking (MN)-based dereplication strategy by UPLC-MS/MS revealed the presence of phospholipids and steroids, while 1H NMR spectroscopy indicated the presence of additional metabolites, such as diketopiperazines (DKPs). Integrated MN and 1H NMR analyses on both the crude and CHCl3 extracts combined with an antibacterial activity-guided isolation approach afforded eight metabolites: a new diketopiperazine, (-)-cyclo(L-trans-Hyp-L-Ile) (8); a known diketopiperazine, cyclo(L-trans-Hyp-L-Phe) (7); two known phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphocholine (1) and 1-O-octadecanoyl-sn-glycero-3-phosphocholine (2); two known steroids, 3β-hydroxycholest-5-ene-7,24-dione (3) and (22E)-3β-hydroxycholesta-5,22-diene-7,24-dione (4); two known monoterpenes, loliolide (5) and 5-epi-loliolide (6). The chemical structures of the isolates were elucidated by a combination of NMR spectroscopy, HRMS and [α]D analyses. All compounds were tested against MRSA and Efm, and compound 3 showed moderate antibacterial activity against MRSA (IC50 value 70 μg/mL). This is the first study that has dealt with chemical and bioactivity profiling of A. sinoxea leading to isolation and characterization of pure sponge metabolites.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094401 ◽

1972 ◽

Vol 30 ◽

pp. 230-231

Author(s):

James Cronshaw ◽

Jamison E. Gilder

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Adenosine Triphosphatase ◽

Higher Plants ◽

High Surface ◽

Root Tip ◽

Animal Cells ◽

Physiological Processes ◽

Tip Cells ◽

Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

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A Scanning Electron Microscopic Study of Pro-Vascular Cellular Morphology in Chaetophora Incressata

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119880 ◽

1985 ◽

Vol 43 ◽

pp. 638-639

Author(s):

A. E. Hotchkiss ◽

A. T. Hotchkiss ◽

R. P. Apkarian

Keyword(s):

Green Algae ◽

Vascular Plants ◽

Vascular System ◽

Higher Plants ◽

Wall Structure ◽

Electron Microscopic ◽

Physiological Processes ◽

Scanning Electron Microscopic Study ◽

Scanning Electron Microscopic ◽

Scanning Electron

Multicellular green algae may be an ancestral form of the vascular plants. These algae exhibit cell wall structure, chlorophyll pigmentation, and physiological processes similar to those of higher plants. The presence of a vascular system which provides water, minerals, and nutrients to remote tissues in higher plants was believed unnecessary for the algae. Among the green algae, the Chaetophorales are complex highly branched forms that might require some means of nutrient transport. The Chaetophorales do possess apical meristematic groups of cells that have growth orientations suggestive of stem and root positions. Branches of Chaetophora incressata were examined by the scanning electron microscope (SEM) for ultrastructural evidence of pro-vascular transport.

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