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Publikationen - Natur- und Wirkstoffchemie

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Preprints

Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.; A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases bioRxiv (2020) DOI: 10.1101/2020.07.22.216432

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.
Forschungsdaten

Stark, P.; Zab, C.; Porzel, A.; Franke, K.; Rizzo, P.; Wessjohann, L. A.; PSYCHE - a valuable experiment in plant NMR-metabolomics RADAR (2020) DOI: 10.22000/338

Dataset: NMR raw dataInstrument: Agilent VNMRS 600 NMR spectrometer
Preprints

Zabel, S.; Brandt, W.; Porzel, A.; Athmer, B.; Kortbeek, R. W. J.; Bleeker, P. M.; Tissier, A.; Two novel 7-epi-zingiberene derivatives with biological activity from Solanum habrochaites are produced by a single cytochrome P450 monooxygenase bioRxiv (2020) DOI: 10.1101/2020.04.21.052571

Secretions from glandular trichomes potentially protect the plant against a variety of aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display substantial growth inhibitory activities against a range of microorganisms, including Bacillus subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.
Publikation

Hussain, H.; Ali, I.; Elizbit, .; Hussain, W.; Mamadalieva, N. Z.; Hussain, A.; Ali, M.; Ahmed, I.; Ullah, I.; Green, I. R.; Synthetic Studies towards Fungal glycosides: An Overview Curr. Org. Chem. 24, 2865-2901, (2020) DOI: 10.2174/1385272824999201105160034

Fungi have provided intriguing chemical diversity and have additionally proven to be a tremendous source for a great variety of therapeutic molecules. Various fungal glycosides have been reported from fungi and the majority of these metabolites possess cytotoxic and antimicrobial effects. Although natural products are obtained in most cases in small amounts from the specific natural source, total syntheses of these valuable commodities remain one of the most important ways of obtaining them on a large scale for more detailed and comprehensive biological studies. In addition, the total synthesis of secondary metabolites is a useful tool, not only for the disclosure of novel complex pharmacologically active molecules but also for the establishment of cutting-edge methodologies in synthetic chemistry. Numerous fungal glycosides have been synthesized in the last four decades regarding the following natural product classes viz., tetramic acid glycosides (epicoccamides A and D), polyketide glycosides (TMC-151C), 2-pyrone glycosides (epipyrone A), diterpene glycosides (sordarin), depside glycosides (CRM646-A and –B, KS-501 and KS- 502), caloporosides (caloporoside A), glycolipids (emmyguyacins A and B, acremomannolipin A), and cerebrosides (cerebroside B, Asperamide B, phalluside-1, Sch II). The current literature review about fungal glycoside synthetic studies is, therefore, of interest for a wide range of scientists and researchers in the field of organic, natural product, and medicinal chemists as it outlines key strategies of fungal glycosides and, in particular, glycosylation, the known biological and pharmacological effects of these natural compounds have afforded a new dimension of exposure.
Publikation

Hussain, W.; Batool, S.; Bahar, S.; Shahida, B.; Bangash, B.; Ali, A.; Hussain, H.; Ali, I.; Ali, M.; Bussmann, R. W.; Ethnopharmacological survey of herbal remedies used in the treatment of hepatitis Ethnobot. Res. App. 20, 1-29, (2020) DOI: 10.32859/era.20.24.1-29

Background: Hepatitis is a serious illness and can be fatal, which has caused a dangerous ratio round the globe especially in the developed and underdeveloping countries. About 1.4 million deaths are reported each year from the different types of hepatitis especially hepatitis B virus (HBV) and hepatitis C virus (HCV) which cause 90% mortality and the other 10% deaths are caused due to all other hepatitis types. Methods: By using different online database such as Google scholar, Science Direct Navigator, ISI Web of Knowledge, Elsevier, springer link, Test databases, research gate and PubMed, date were retrieved by giving different keywords. Various keywords were used on plants used traditionally to treat hepatitis. Results: A total of 128 medicinal plant species were identified, belonging to 108 genera and 61 botanical families by reviewing 220 research articles. From the study, it showed that the medicinal plants are used from the very ancient time to cure hepatitis worldwide. These medicinal plants are used in different parts of the world as a traditional herbal medicine for the control of hepatitis. Conclusion: Our literature review will help the scientific communities to identify anti-hepatitis plant in order to isolate novel anti-hepatitis natural products.
Publikation

Hussain, H.; Ali, I.; Wang, D.; Mamadalieva, N. Z.; Hussain, W.; Csuk, R.; Loesche, A.; Fischer, L.; Staerk, D.; Anam, S.; AlZain, M. N.; Mushtaq, M.; Ul-Haq, Z.; Ullah, R.; Noman, O. M.; Abbas, G.; Green, I. R.; 4-Benzyloxylonchocarpin and Muracatanes A-C from Ranunculus muricatus L. and Their Biological Effects Biomolecules 10, 1562, (2020) DOI: 10.3390/biom10111562

Ranunculus muricatus L. is a spiny fruit buttercup that is used in various traditional medicinal systems. In the current investigation of R. muricatus, the new chalcone 4-benzyloxylonchocarpin (1), the new anthraquinone muracatanes A (2), the new-to-nature anthraquinone muracatanes B (3), and the new naphthalene analog muracatanes C (4) were isolated, in addition to the three previously reported compounds, 4-methoxylonchocarpin (5), β-sitosterol (6), and β-sitosterol β-D-glucopyranoside (7). Their structures were elucidated using 1D (1H and 13C) and 2D (COSY, HSQC, and HMBC) NMR spectroscopy and HR-ESI-MS. Chalcone 1 showed potent acetylcholinesterase inhibitory effects with Ki of 5.39 µM and Ki′ of 3.54 µM, but none of the isolated compounds showed inhibitory activity towards butyrylcholinesterase. Anthraquinone 3 illustrated α-glucosidase inhibitory effects with IC50-values of 164.46 ± 83.04 µM. Compound 5 displayed moderate cytotoxic activity towards ovarian carcinoma (A2780, IC50 = 25.4 µM), colorectal adenocarcinoma (HT29, IC50 = 20.2 µM), breast cancer (MCF7, IC50 = 23.7 µM), and thyroid carcinoma (SW1736, IC50 = 26.2 µM) while it was inactive towards pharynx carcinoma (FaDu: IC50 > 30 µM).
Publikation

Humpierre, A. R.; Zanuy, A.; Saenz, M.; Garrido, R.; Vasco, A. V.; Pérez-Nicado, R.; Soroa-Milán, Y.; Santana-Mederos, D.; Westermann, B.; Vérez-Bencomo, V.; Méndez, Y.; García-Rivera, D.; Rivera, D. G.; Expanding the Scope of Ugi Multicomponent Bioconjugation to Produce Pneumococcal Multivalent Glycoconjugates as Vaccine Candidates Bioconjugate Chem. 31, 2231-2240, (2020) DOI: 10.1021/acs.bioconjchem.0c00423

Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide–protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.
Publikation

Holzmeyer, L.; Hartig, A.-K.; Franke, K.; Brandt, W.; Muellner-Riehl, A. N.; Wessjohann, L. A.; Schnitzler, J.; Evaluation of plant sources for antiinfective lead compound discovery by correlating phylogenetic, spatial, and bioactivity data Proc. Natl. Acad. Sci. U.S.A. 117, 12444-12451, (2020) DOI: 10.1073/pnas.1915277117

The continued high rates of using antibiotics in healthcare and livestock, without sufficient new compounds reaching the market, has led to a dramatic increase in antimicrobial resistance, with multidrug-resistant bacteria emerging as a major public health threat worldwide. Because the vast majority of antiinfectives are natural products or have originated from them, we assessed the predictive power of plant molecular phylogenies and species distribution modeling in the search for clades and areas which promise to provide a higher probability of delivering new antiinfective compound leads. Our approach enables taxonomically and spatially targeted bioprospecting and supports the battle against the global antimicrobial crisis.
Publikation

Guerra, T.; Schilling, S.; Hake, K.; Gorzolka, K.; Sylvester, F.-P.; Conrads, B.; Westermann, B.; Romeis, T.; Calcium‐dependent protein kinase 5 links calcium‐signaling with N‐Hydroxy‐L‐pipecolic acid‐ and SARD1‐dependent immune memory in systemic acquired resistance New Phytol. 225, 310-325, (2020) DOI: 10.1111/nph.16147

Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self‐maintaining phytohormone salicylic acid (SA)‐defense loop.In spatial and temporal resolution, we show that calcium‐dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR‐inducing metabolite N‐hydroxy‐L‐pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1)Plants of increased CPK5, but not CPK6, signaling display an ‘enhanced SAR’ phenotype towards a secondary bacterial infection. In the sard1‐1 background, CPK5‐mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost.The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca2+], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity.
Publikation

Gladchuk, A.; Shumilina, J.; Kusnetsova, A.; Bureiko, K.; Billig, S.; Tsarev, A.; Alexandrova, I.; Leonova, L.; Zhukov, V. A.; Tikhonovich, I. A.; Birkemeyer, C.; Podolskaya, E.; Frolov, A.; High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Methods Protoc. 3, 36, (2020) DOI: 10.3390/mps3020036

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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