Publications by authors named "Oliver Kayser"

89 Publications

In Vitro Production and Exudation of 20-Hydroxymaytenin from (Eckl. and Zeyh.) Loes. Cell Culture.

Plants (Basel) 2021 Jul 21;10(8). Epub 2021 Jul 21.

Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund University, 44227 Dortmund, Germany.

The metabolite 20-Hydroxymaytenin (20-HM) is a member of the quinone-methide pentacyclic triterpenoids (QMTs) group. This metabolite group is present only in Celastraceae plants, and it has shown various biological activities from antioxidant to anticancer properties. However, most QMTs metabolites including 20-HM cannot be synthesized in a laboratory. Therefore, we optimized a plant tissue culture protocol and examined the potential of (synonym. ) to produce 20-HM in an in vitro experiment. For the first time, we reported the optimum callus induction medium with a high percentage success rate of 82% from the combination of 1 mg/L indole-3-butyric acid and 5 mg/L naphthalene acetic acid. Later, our cell suspension culture cultivated in the optimum medium provided approximately 0.35 mg/g fresh weight of 20-HM. This concentration is roughly 87.5 times higher than a concentration of 20-HM presenting in (Celastraceae) leaves. In addition, we also found that 20-HM presented in a cultivation medium, suggesting that cells secreted 20-HM as an exudate in our experiment. Noticeably, 20-HM was missing when cf. occurred in the medium. These findings hint at an antifungal property of 20-HM.
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http://dx.doi.org/10.3390/plants10081493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8398937PMC
July 2021

In vitro metabolism of tebuconazole, flurtamone, fenhexamid, metalaxyl-M and spirodiclofen in Cannabis sativa L. (hemp) callus cultures.

Pest Manag Sci 2021 Jul 26. Epub 2021 Jul 26.

Faculty of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, TU Dortmund University, Dortmund, Germany.

Background: Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail.

Results: With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 μm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21.

Conclusion: The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa.
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http://dx.doi.org/10.1002/ps.6575DOI Listing
July 2021

Evaluation of Callus Cultures to Elucidate the Metabolism of Tebuconazole, Flurtamone, Fenhexamid, and Metalaxyl-M in L., (L.) Merr., L., and L.

J Agric Food Chem 2020 Nov 20. Epub 2020 Nov 20.

Faculty of Biochemical and Chemical Engineering Chair of Technical Biochemistry, TU Dortmund University, Emil-Figge-Strasse 66, 44227 Dortmund, Germany.

Plant cell cultures can be used to identify the metabolic degradation of pesticides in crops. Therefore, L., (L.) Merr., L. and L. were used to elucidate the metabolic degradation of the following pesticides: tebuconazole, flurtamone, fenhexamid, and metalaxyl-M. Callus cultures were treated with 10 μM of the named pesticides by passive diffusion out of the nutrition agar while young plants were hydroponically exposed to it. After 14 days, the comparison of and experiments showed that the metabolic degradation is well described by callus cultures. The intracellular uptake of all pesticides and a broad spectrum of exemplarily hydroxylated and conjugated metabolites were detectable. Overall, the comparability of the nature of residues out of both experiments with the regulatory guideline metabolism studies could be demonstrated. Therefore, we recommend it as a potential screening tool to elucidate the metabolism of pesticides in crops.
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http://dx.doi.org/10.1021/acs.jafc.0c05277DOI Listing
November 2020

Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast.

Appl Microbiol Biotechnol 2020 Nov 12;104(22):9551-9563. Epub 2020 Oct 12.

TU Dortmund University, Technical Biochemistry, Emil-Figge-Strasse 66, 44227, Dortmund, Germany.

Heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa.Key points• First time critical review of the heterologous process for recombinant THCA/CBDA production and critical review of bottlenecks and limitations for a bioengineered technical process• Integrative approach of protein engineering, systems biotechnology, and biochemistry of yeast physiology and biosynthetic cannabinoid enzymes• Comparison of NphB and CsPT aromatic prenyltransferases as rate-limiting catalytic steps towards cannabinoids in yeast as platform organisms Graphical abstract.
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http://dx.doi.org/10.1007/s00253-020-10798-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595985PMC
November 2020

Corrigendum to "Ozonation of rivaroxaban production waste water and comparison of generated transformation products with known in vivo and in vitro metabolites" [Sci. Total Environ. 714 (2020), 136825].

Sci Total Environ 2020 Nov 21;742:140865. Epub 2020 Jul 21.

Department of Technical Biochemistry, Technical University of Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany.

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http://dx.doi.org/10.1016/j.scitotenv.2020.140865DOI Listing
November 2020

Editorial: Biotechnological Production and Conversion of Aromatic Compounds and Natural Products.

Front Bioeng Biotechnol 2020 19;8:646. Epub 2020 Jun 19.

Department of Technical Biochemistry, Technical University of Dortmund, Dortmund, Germany.

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http://dx.doi.org/10.3389/fbioe.2020.00646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318798PMC
June 2020

Editorial.

Planta Med 2020 05 26;86(8):e3. Epub 2020 May 26.

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http://dx.doi.org/10.1055/a-1182-5301DOI Listing
May 2020

Challenges at the Time of COVID-19: Opportunities and Innovations in Antivirals from Nature.

Planta Med 2020 Jul 20;86(10):659-664. Epub 2020 May 20.

University Medicine Rostock, Chair of Complementary Medicine, Rostock, Germany.

As viral infections are an increasing threat to human societies, the need for new therapeutic strategies is becoming even more obvious. As no vaccine is available for COVID-19, the development of directly acting antiviral agents and preventive strategies have to be considered. Nature provides a huge reservoir of anti-infectious compounds, from which we can deduce innovative ideas, therapies, and products. Anti-adhesive natural products interact with the receptor-mediated recognition and early interaction of viruses with the host cells, leading to a reduced internalisation of the virus and reduced infections (e.g., procyanidin-B-2-di-O-gallate against influenza and herpes virus). Lignans like podophyllotoxin and bicyclol show strong antiviral activities against different viruses, and essential oils can directly interact with viral membranes and reduce the host's inflammatory responses (e.g., 1,8-cineol). extracts stimulate the immune system, and bioavailable alkamides are key players by interacting with immunomodulating cannabinoid receptors. COVID-19 and SARS-CoV-2 infections have, in part, successfully been treated in China by preparations from traditional Chinese medicine and, while it is too early to draw conclusions, some promising data are available. There is huge potential, but intensified research is needed to develop evidence-based medicines with a clearly defined chemical profile. Intensified research and development, and therefore funding, are needed for exploiting nature's reservoir against viral infections. Combined action for basic research, chemistry, pharmacognosy, virology, and clinical studies, but also supply chain, sustainable sourcing, and economic aspects have to be considered. This review calls for intensified innovative science on natural products for the patients and for a healthier world!
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http://dx.doi.org/10.1055/a-1177-4396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356065PMC
July 2020

Ozonation of rivaroxaban production waste water and comparison of generated transformation products with known in vivo and in vitro metabolites.

Sci Total Environ 2020 Apr 21;714:136825. Epub 2020 Jan 21.

Department of Technical Biochemistry, Technical University of Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany.

During production of rivaroxaban, an orally administrated anticoagulant, process waste water is generated at several stages of manufacturing. Due to low biodegradability in conventional waste water treatment plants, it is currently incinerated. Thus, new pre-treatment methods for rivaroxaban-containing waste water could facilitate its subsequent biological processing. In this study, ozonation was investigated as the pre-treatment method, with special emphasis on the elimination of the target compound and the generation of its main transformation products. These were determined by high performance liquid chromatography - high resolution mass spectrometry (HPLC-HRMS). Our results show that the concentration of rivaroxaban in the ozonated waste water can be effectively reduced (below detection limit) under acidic conditions, whereas neutral or basic reaction environment proved less suitable. Four main transformation products were identified and their concentrations determined. Rivaroxaban and its most prominent transformation product acetoxamide were elucidated in multiple-stage mass spectrometry (MS) experiments. Transformation products derived from the degradation of rivaroxaban were then compared to its natural (animal and human) metabolites as well as other known derivatives. In contrast to previously published rivaroxaban-derived metabolites, the presented ozonation experiments resulted in new transformation products.
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http://dx.doi.org/10.1016/j.scitotenv.2020.136825DOI Listing
April 2020

Virus-induced gene silencing (VIGS) in L.

Plant Methods 2019 26;15:157. Epub 2019 Dec 26.

Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.

Background: The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable.

Results: For this, a computational approach was employed to analyze the L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the () and (), were identified in Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach Here we show for the first time a gene knockdown in using the () in a silencing vector system. Plants transiently transformed with the strain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The successful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of transcripts from 70 to 73% for and , respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the -based vector system.

Conclusions: The applied VIGS-method can be used for reverse genetic studies in to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between available genomic data and biochemical information of this important medicinal plant.
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http://dx.doi.org/10.1186/s13007-019-0542-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931244PMC
December 2019

Secondary metabolites from isolated from .

Nat Prod Res 2021 Jul 7;35(14):2324-2328. Epub 2019 Oct 7.

Natural Products Research Group, Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Indonesia.

Fractionation and purification of the ethyl acetate extract of , an endophytic fungus from the leaves of , yielded one new compound, diaporthindoic acid (), along with seven known compounds (-). The new compound was characterized and established by the basis of extensive spectroscopic methods including NMR (1D and 2D) and HRMS. Compound showed the best citotoxicity against murine leukemia P-388 cells with an IC value of 0.41 g/mL. All compounds (-) were also tested for their antimicrobial activities. To the best of our knowledge, this is the first chemical evaluation of fungal derived from .
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http://dx.doi.org/10.1080/14786419.2019.1672685DOI Listing
July 2021

Best practice in research - Overcoming common challenges in phytopharmacological research.

J Ethnopharmacol 2020 Jan 14;246:112230. Epub 2019 Sep 14.

Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa; SAMRC Herbal Drugs Research Unit, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa. Electronic address:

Background: The pharmacology, toxicology and pharmacokinetics of bioactive preparations derived from natural sources has become a flourishing field of research. However, researching complex extracts and natural products faces numerous challenges. More broadly in recent years the critique of pharmacological research, and specifically its design, the methods used and reporting has intensified.

Aims: This consensus document provides a perspective on what constitutes best practice in pharmacological research on bioactive preparations derived from natural sources, providing a perspective of what the leading specialist journals in the field consider as the core characteristics of good research.

Approach ('methods'): The editors-in-chief of seven journals developed this best practice statement in an iterative process. A first draft of the guidelines (prepared by MH) was then discussed and amended by the other editors.

Outcomes: Core to this contribution is a table which provides detailed advice including simple points like a use of appropriate controls and the full taxonomic validity of the material under investigation (see also below), to the relevance of the model for the question being researched (e.g., can specific in silico or in vitro models really address the species anti-inflammatory activity?). Therefore, obviously, researchers must pay detailed attention to reporting and discussing such studies. This information must be discussed critically (as much as it is possible based on the published papers) in terms of their scientific quality and validity. While these points are obvious, as editors, we are aware that they are often not properly implemented.

Conclusion: We call for an approach which incorporates a careful design, meticulous execution and a detailed reporting of studies focusing on the pharmacology/bioactivity of bioactive preparations. Clearly testable research questions must be developed and investigated experimentally. As the founder of pharmacology Claude Bernard put it already in 1865: '…. either the experimenter's hypothesis will be disproved or it will be proved by experiment. When experiment disproves its preconceived ideas, the experimenter must discard or modify it.'
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http://dx.doi.org/10.1016/j.jep.2019.112230DOI Listing
January 2020

Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes.

Plant Sci 2019 Jul 12;284:108-116. Epub 2019 Apr 12.

Department of Technical Biochemistry, Technical University Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany. Electronic address:

Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.
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http://dx.doi.org/10.1016/j.plantsci.2019.04.008DOI Listing
July 2019

Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production.

Molecules 2019 Feb 22;24(4). Epub 2019 Feb 22.

Technical Biochemistry, Department of Biochemical and Chemical Engineering, Technical University Dortmund, D-44227 Dortmund, Germany.

Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.
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http://dx.doi.org/10.3390/molecules24040796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412926PMC
February 2019

Editorial.

Planta Med 2019 Mar 26;85(4):273. Epub 2019 Feb 26.

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http://dx.doi.org/10.1055/a-0832-5297DOI Listing
March 2019

Editorial.

Planta Med 2019 Jan 22;85(2):93. Epub 2019 Jan 22.

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http://dx.doi.org/10.1055/a-0819-4096DOI Listing
January 2019

Chemical composition and biological activity of the essential oil from the root of Courb. native to Saudi Arabia.

Saudi Pharm J 2019 Jan 11;27(1):88-95. Epub 2018 Sep 11.

TU Dortmund University, Technical Biochemistry, Emil-Figge-Strasse 66, D-44227 Dortmund, Germany.

The chemical composition of the essential oil from roots was determined via GC-FID. There were 80 compounds, representing 99.99% of the total oil constituents. Among these, 77.31% were sesquiterpenes, 14.62% were fatty acids, 7.21% were other components (i.e., phenolics, hydrocarbons, etc.), and 0.85% were monoterpenes. The major compounds in the oil were γ-eudesmol (35.31%), 5-guaien-11-ol (14.43%), -cedrol (8.19%), oleic acid (5.23%), bulnesol (4.45%), α-linoleic acid (4.20%), 3,4-dimethoxycinnamic acid (3.83%), palmitic acid (2.69%), isolongifolanone (2.68%), eicosane (1.41%), and cedrol (1.14%). Oxygenated sesquiterpenes were found to represent more than 50% percent of the total oil content. Moreover, the essential oil was evaluated for anti-inflammatory, antioxidant, antipyretic, and antinociceptive activities using and models. Additionally, the antioxidant potential of the oil was evaluated using various  antioxidant tests, including DPPH, ABTS and FRAP. At a dose of 240 µl/kg, the oil showed anti-inflammatory (59.12%), antipyretic (37.00 ± 0.11), and antinociceptive (47.58%) activities and showed significant (p < 0.001) effect as compared to a standard drug (phenylbutazone and indomethacin). These findings demonstrated that the essential oil of root could be used as a natural source for their anti-inflammatory, antinociceptive, antipyretic, and antioxidant effects.
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http://dx.doi.org/10.1016/j.jsps.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323148PMC
January 2019

Traditional use of ethnomedicinal native plants in the Kingdom of Saudi Arabia.

J Ethnobiol Ethnomed 2019 Jan 9;15(1). Epub 2019 Jan 9.

Technical Biochemistry, TU Dortmund University, Emil-Figge-Strasse 66, 44227, Dortmund, Germany.

The Arabian Peninsula is recognized as an arid area dominated by deserts and poor biodiversity. However, the Kingdom of Saudi Arabia (henceforth abbreviated into KSA) has a wide range of flora, consisting of different species of trees, herbs, and shrubs and containing numerous edible and medicinal plants. The KSA is characterized by its vast area of diverse geographical landscapes and climates. Consequently, there is enormous variation in the distribution of plants across the Kingdom. The traditional use of ethnomedical plants in the KSA represents a strong interconnection among familiar remedies, health, diet, and traditional healing practices characterized by specific cultures. The present paper reviews a collection of medicinal plants in KSA used in ethnomedicine. This review might be useful in developing strategies for the sustainable use of medicinal plants which are among the threatened important natural resources in folk medicine in the KSA. The present study reports 309 genera which cover 471 species from a total of 2253 known species belonging to 89 families. The most dominating families are Asteraceae, Fabaceae, Lamiaceae, Euphorbiaceae, Solanaceae, Apiaceae, Brassicaceae, Chenopodiaceae, Poaceae, Amaranthaceae, Boraginaceae, Apocynaceae, Convolvoulaceae, Asclepiadaceae, Capparaceae, Polygonaceae, and Zygophyllaceae.
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http://dx.doi.org/10.1186/s13002-018-0263-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325684PMC
January 2019

Editorial.

Planta Med 2019 Jan 3;85(1). Epub 2019 Jan 3.

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http://dx.doi.org/10.1055/a-0774-4019DOI Listing
January 2019

Chemical fingerprinting of single glandular trichomes of Cannabis sativa by Coherent anti-Stokes Raman scattering (CARS) microscopy.

BMC Plant Biol 2018 Nov 12;18(1):275. Epub 2018 Nov 12.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, 44227, Dortmund, Germany.

Background: Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type.

Results: Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures.

Conclusion: Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.
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http://dx.doi.org/10.1186/s12870-018-1481-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233497PMC
November 2018

The Phytochemical and Biological Investigation of Root Native to the Kingdom of Saudi Arabia.

Molecules 2018 Jul 28;23(8). Epub 2018 Jul 28.

Department of Pharmacognosy, Faculty of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.

Extensive phytochemical analysis of different root fractions of Courb. (Euphorbiaceae) has resulted in the isolation and identification of 22 secondary metabolites. 6-hydroxy-8-methoxycoumarin-7--β-d-glycopyranoside () and 2-hydroxymethyl -methyltryptamine () were isolated and identified as new compounds along with the known diterpenoid (, , , and ), triterpenoid ( and ), flavonoid (, , , , and ), coumarinolignan (⁻), coumarin (), pyrimidine (), indole (, ), and tyramine-derived molecules (⁻). The anti-inflammatory, analgesic, and antipyretic activities were evaluated for fifteen of the adequately available isolated compounds (⁻, ⁻, , , , , and ). Seven (, , , , , and of the tested compounds showed a significant analgesic effect ranging from 40% to 80% at 10 mg/kg in two in vivo models. Compound could also prove its analgesic property (67.21%) when it was evaluated on a third in vivo model at the same dose. The in vitro anti-inflammatory activity was also recorded where all compounds showed the ability to scavenge nitric oxide (NO) radical in a dose-dependent manner. However, eight compounds (, , , , , , , and ) out of the fifteen tested compounds exhibited considerable in vivo anti-inflammatory activity which reached 64.91% for compound at a dose of 10 mg/kg. Moreover, the tested compounds exhibited an antipyretic effect in a yeast-induced hyperthermia in mice. The activity was found to be highly pronounced with compounds , , , , , and which decreased the rectal temperature to about 37 °C after 2 h of the induced hyperthermia (~39 °C) at a dose of 10 mg/kg. This study could provide scientific evidence for the traditional use of as an anti-inflammatory, analgesic, and antipyretic.
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http://dx.doi.org/10.3390/molecules23081892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222854PMC
July 2018

Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativaL.

J Biotechnol 2018 Oct 24;284:17-26. Epub 2018 Jul 24.

Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany.

Cannabinoids are secondary natural products from the plant Cannabis sativaL. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the Δ-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - Δ-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the C-terminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52 °C to 57 °C. We generated two CBDAS variants, C_S116A and C_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production.
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http://dx.doi.org/10.1016/j.jbiotec.2018.07.031DOI Listing
October 2018

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in .

Front Plant Sci 2018 9;9:537. Epub 2018 May 9.

Department of Technical Biochemistry, TU Dortmund University, Dortmund, Germany.

The liverwort belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, assembly and annotation of transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in . Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in . In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for . The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the family.
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http://dx.doi.org/10.3389/fpls.2018.00537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954354PMC
May 2018

Ethnobotany and Medicinal Plant Biotechnology: From Tradition to Modern Aspects of Drug Development.

Authors:
Oliver Kayser

Planta Med 2018 Aug 24;84(12-13):834-838. Epub 2018 May 24.

TU Dortmund University, Biochemical and Chemical Engineering, Technical Biochemistry, Dortmund, Germany.

Secondary natural products from plants are important drug leads for the development of new drug candidates for rational clinical therapy and exhibit a variety of biological activities in experimental pharmacology and serve as structural template in medicinal chemistry. The exploration of plants and discovery of natural compounds based on ethnopharmacology in combination with high sophisticated analytics is still today an important drug discovery to characterize and validate potential leads. Due to structural complexity, low abundance in biological material, and high costs in chemical synthesis, alternative ways in production like plant cell cultures, heterologous biosynthesis, and synthetic biotechnology are applied. The basis for any biotechnological process is deep knowledge in genetic regulation of pathways and protein expression with regard to todays "omics" technologies. The high number genetic techniques allowed the implementation of combinatorial biosynthesis and wide genome sequencing. Consequently, genetics allowed functional expression of biosynthetic cascades from plants and to reconstitute low-performing pathways in more productive heterologous microorganisms. Thus, biosynthesis in heterologous hosts requires fundamental understanding of pathway reconstruction and multitude of genes in a foreign organism. Here, actual concepts and strategies are discussed for pathway reconstruction and genome sequencing techniques cloning tools to bridge the gap between ethnopharmaceutical drug discovery to industrial biotechnology.
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http://dx.doi.org/10.1055/a-0631-3876DOI Listing
August 2018

Subcellular localization defines modification and production of Δ-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.

Biotechnol Lett 2018 Jun 4;40(6):981-987. Epub 2018 Apr 4.

Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Schnittspahnstraße 4, 64287, Darmstadt, Germany.

Objective: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis.

Results: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g activity towards CBCA production).

Conclusion: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.
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http://dx.doi.org/10.1007/s10529-018-2545-0DOI Listing
June 2018

Optimization of Δ-tetrahydrocannabinolic acid synthase production in Komagataella phaffii via post-translational bottleneck identification.

J Biotechnol 2018 Apr 13;272-273:40-47. Epub 2018 Mar 13.

Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany. Electronic address:

Δ-Tetrahydrocannabinolic acid (THCA) is a secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain or reducing spasticity associated with multiple sclerosis. Here, we investigated the influence of the co-expression of 12 helper protein genes from Komagataella phaffii (formerly Pichia pastoris) on the functional expression of the Δ-tetrahydrocannabinolic acid synthase (THCAS) heterologously expressed in K. phaffii by screening 21 clones of each transformation. Our findings substantiate the necessity of a suitable screening system when interfering with the secretory network of K. phaffii. We found that co-production of the chaperones CNE1p and Kar2p, the foldase PDI1p, the UPR-activator Hac1p as well as the FAD synthetase FAD1p enhanced THCAS activity levels within the K. phaffii cells. The strongest influence showed co-expression of Hac1s - increasing the volumetric THCAS activities 4.1-fold on average. We also combined co-production of Hac1p with the other beneficial helper proteins to further enhance THCAS activity levels. An optimized strain overexpressing Hac1s, FAD1 and CNE1 was isolated that showed 20-fold increased volumetric, intracellular THCAS activity compared to the starting strain. We used this strain for a whole cell bioconversion of cannabigerolic acid (CBGA) to THCA. After 8 h of incubation at 37 °C, the cells produced 3.05 g L THCA corresponding to 12.5% g g.
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http://dx.doi.org/10.1016/j.jbiotec.2018.03.008DOI Listing
April 2018

Current Perspectives on Biotechnological Cannabinoid Production in Plants.

Planta Med 2018 Mar 4;84(4):214-220. Epub 2018 Jan 4.

Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.

The plant contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other -derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on cell culture systems, propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated.
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http://dx.doi.org/10.1055/s-0043-125087DOI Listing
March 2018

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Plant Cell Physiol 2018 Jan;59(1):107-118

Technical Biochemistry, Department of Biochemical and Chemical Engineering, Technical University Dortmund, D-44227 Dortmund, Germany.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6β-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.
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http://dx.doi.org/10.1093/pcp/pcx165DOI Listing
January 2018

Ozone pretreatment of process waste water generated in course of fluoroquinolone production.

Chemosphere 2017 Oct 12;185:953-963. Epub 2017 Jul 12.

Department of Technical Biochemistry, Technical University of Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany. Electronic address:

During production of active pharmaceutical ingredients, process waste water is generated at several stages of manufacturing. Whenever possible, the resulting waste water will be processed by conventional waste water treatment plants. Currently, incineration of the process waste water is the method to eliminate compounds with high biological activity. Thus, ozone treatment followed by biological waste water treatment was tested as an alternative method. Two prominent representatives of the large group of fluoroquinolone antibiotics (ciprofloxacin and moxifloxacin) were investigated, focussing on waste water of the bulk production. Elimination of the target compounds and generation of their main transformation products were determined by liquid chromatography - high resolution mass spectrometry (LC-HRMS). The obtained results demonstrated, that the concentration of moxifloxacin and its metabolites can be effectively reduced (>99.7%) prior entering the receiving water. On the contrary, the concentration of ciprofloxacin and its metabolites remained too high for safe discharge, necessitating application of prolonged ozonation for its further degradation. The required ozonation time can be estimated based on the determined kinetics. To assure a low biological activity the ecotoxicity of the ozonated waste water was investigated using three trophic levels. By means of multiple-stage mass spectrometry (MS) experiments several new transformation products of the fluoroquinolones were identified. Thus, previously published proposed structures could be corrected or confirmed.
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http://dx.doi.org/10.1016/j.chemosphere.2017.07.040DOI Listing
October 2017

Engineering yeasts as platform organisms for cannabinoid biosynthesis.

J Biotechnol 2017 Oct 8;259:204-212. Epub 2017 Jul 8.

Department of Technical Biochemistry, TU Dortmund University, Emil-Figge Str. 66, 44227 Dortmund, Germany.

Δ-tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis satival. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme's coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform.
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http://dx.doi.org/10.1016/j.jbiotec.2017.07.008DOI Listing
October 2017
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