Die Tetrahydroisochinolin-Alkaloide bilden eine groûe Familie von mehr als 2500 [1] bekannten, me... more Die Tetrahydroisochinolin-Alkaloide bilden eine groûe Familie von mehr als 2500 [1] bekannten, meist pharmakologisch bedeutsamen pflanzlichen Sekundärmetaboliten ± von einfachen 1-Alkylisochinolinen wie Anhalonidin 1 [2] (Schema 1) zu komplexen polycyclischen Benzylisochinolinen wie Schema 1. Bekannter [2] biogenetischer Ursprung von Tetrahydroisochinolinen wie Anhalonidin über Pictet-Spengler-Kondensation und der vorgeschlagene neue Weg zu Dioncophyllin A 2.
Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. ... more Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots. Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 % for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.
Although vertical column, flat-plate, tubular, bubble column and airlift photobioreactors are wid... more Although vertical column, flat-plate, tubular, bubble column and airlift photobioreactors are widely used for the cultivation of diatoms, it has been shown that bubble-induced hydrodynamic stress created in these types of bioreactor can damage cells. Therefore, three single-use surface-aerated bag bioreactors, known for their outstanding results in cultivating shearsensitive mammalian cells, were for the first time investigated for their suitability in growing the model microalgae Phaeodactylum tricornutum. All of the systems, which were additionally aerated with CO 2 and equipped with illumination systems providing different light qualities, guaranteed a 22-to 43-fold increase in cell density within seven days, without any addition of cell protection agents or changes in cell morphology. Maximum cell density and dry biomass were achieved in the orbitally shaken 2D-bag by using cool-white fluorescent tubes.
The structure of antidesmone, an alkaloid from Antidesma membranaceum Müll. Arg. and A. venosum E... more The structure of antidesmone, an alkaloid from Antidesma membranaceum Müll. Arg. and A. venosum E. Mey. (Euphorbiaceae), was revised to be (S)-4,8-dioxo-3-methoxy-2-methyl-5-n-octyl-1,4,5,6,7,8-hexahydroquinoline [(S)-2], not the isoquinoline derivative 1, as assumed previously. The revision was initiated by biosynthetic feeding experiments of one of our groups.
An efficient evaluation procedure for the chemical screening and on-line structural elucidation o... more An efficient evaluation procedure for the chemical screening and on-line structural elucidation of dimeric naphthylisoquinoline alkaloids has been developed. The method is based on the lead tetraacetate oxidation of the central binaphthalene core of the alkaloids. UV spectra of the extracts after addition of the oxidant show, in the presence of naphthylisoquinoline dimers, the appearance of a characteristic long-wavelength absorption indicative of dinaphthoquinones. The efficiency and relevance of the method has been demonstrated in the discovery of a constitutionally and configurationally new dimeric naphthylisoquinoline alkaloid, named ancistrogriffithine A (4), from the previously uninvestigated Asian liana Ancistrocladus griffithii. After verification of this screening result by LC-ESI-MS/MS, the constitution and the relative configuration of the compound were elucidated on line, by LC-NMR and LC-CD on the extract. Using an LC-NMR-WET-ROESY experiment, it was possible for the first time to determine the relative axial configuration of a natural biaryl compound on line, by observing long-range ROE interactions. Finally, an oxidative degradation right on the extract delivered the absolute configuration of 4, without isolation of the alkaloid. Ancistrogriffithine A is the as yet only dimeric naphthylisoquinoline from an Asian Ancistrocladaceae plant.
The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/... more The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/5 Linsmaier and Skoog medium was studied. During a lag phase in growth, droserone concentrations in the medium reached a value of 2.1 mg g À1 fr. wt. After this maximum value the concentration decreased slightly to 1.8 mg g À1 fr. wt., while the growth of the calli was enhanced (25% increase in fr. wt. within 7 days). Plumbagin and isoshinanolone were likewise present in the medium. By feeding 13 C 2-labelled acetate to the cultures the biosynthesis of droserone was elucidated. The incorporation of whole C 2-units unambiguously shows its acetogenic origin and ®ts well in the biosynthetic scheme suggested for the structurally Ð and biogenetically Ð related naphthylisoquinoline alkaloids.
European Food Research and Technology, Apr 30, 2023
The so-called "craft beer revolution" has increased the demand for new styles of beers, often wit... more The so-called "craft beer revolution" has increased the demand for new styles of beers, often with new ingredients like flavour extracts. In recent years, synthetic biology has realized the production of a plethora of plant secondary metabolites in microbial hosts, which could provide an alternative source for these compounds. In this study, we selected a in situ flavour production approach for grape flavour addition. We used an O-methyl anthranilate (OmANT) producing laboratory Saccharomyces cerevisiae strain in co-fermentations with an industrial beer yeast strain WLP644. The laboratory strain provided an ease of genetic manipulation and the desirable properties of the WLP644 strain were not modified in this approach. In shake flasks, a 10:90 ratio of the yeasts produced grape flavoured beer with the yeast produced flavour compound in a range normally used for flavoured beverages. Hopped and unhopped beers were analysed by VTT's trained sensory panel and with olfactory GC-MS. OmANT was successfully detected from the beers as a floral odour and flavour. Moreover, no off-flavours were detected and aroma profiles outside the grape flavour were rather similar. These results indicate that the co-fermentation principle is a suitable approach to change the flavour profiles of beers with a simple yeast strain drop-in approach.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Plants are capable of producing an overwhelming variety of secondary metabolites, both in terms o... more Plants are capable of producing an overwhelming variety of secondary metabolites, both in terms of complexity and quantity. Yet, when compared with the situation in microorganisms this impressive metabolic machinery is hardly exploited. One of the main reasons is the limited molecular insight into plant secondary metabolism, which hampers the design of effective metabolic engineering strategies. To address these shortcomings we employ a functional genomics based technology platform that enables comprehensive investigations and large-scale gene discovery programs in plant metabolism. The platform relies primarily on the integration of genome-wide transcript profiling with metabolite profiling and is applicable to any plant species or system to map the biosynthesis of any metabolite.
In this paper, an analytical method for the analysis of molecular lipids in algae samples is repo... more In this paper, an analytical method for the analysis of molecular lipids in algae samples is reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh performance liquid chromatography combined with mass spectrometry (UPLC-MS). For further characterization of lipids, MS/MS analyses are carried out utilizing either a separate instrument (e.g., LTQ-Orbitrap mass spectrometer) or simultaneous fragmentation with the same instrument. Throughput of the method is over 100 samples/d. The repeatability is good, and the relative standard deviation of spiked samples is <15%.
Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described.... more Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described. Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids, and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.
Die Tetrahydroisochinolin-Alkaloide bilden eine groûe Familie von mehr als 2500 [1] bekannten, me... more Die Tetrahydroisochinolin-Alkaloide bilden eine groûe Familie von mehr als 2500 [1] bekannten, meist pharmakologisch bedeutsamen pflanzlichen Sekundärmetaboliten ± von einfachen 1-Alkylisochinolinen wie Anhalonidin 1 [2] (Schema 1) zu komplexen polycyclischen Benzylisochinolinen wie Schema 1. Bekannter [2] biogenetischer Ursprung von Tetrahydroisochinolinen wie Anhalonidin über Pictet-Spengler-Kondensation und der vorgeschlagene neue Weg zu Dioncophyllin A 2.
Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. ... more Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots. Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83 % for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.
Although vertical column, flat-plate, tubular, bubble column and airlift photobioreactors are wid... more Although vertical column, flat-plate, tubular, bubble column and airlift photobioreactors are widely used for the cultivation of diatoms, it has been shown that bubble-induced hydrodynamic stress created in these types of bioreactor can damage cells. Therefore, three single-use surface-aerated bag bioreactors, known for their outstanding results in cultivating shearsensitive mammalian cells, were for the first time investigated for their suitability in growing the model microalgae Phaeodactylum tricornutum. All of the systems, which were additionally aerated with CO 2 and equipped with illumination systems providing different light qualities, guaranteed a 22-to 43-fold increase in cell density within seven days, without any addition of cell protection agents or changes in cell morphology. Maximum cell density and dry biomass were achieved in the orbitally shaken 2D-bag by using cool-white fluorescent tubes.
The structure of antidesmone, an alkaloid from Antidesma membranaceum Müll. Arg. and A. venosum E... more The structure of antidesmone, an alkaloid from Antidesma membranaceum Müll. Arg. and A. venosum E. Mey. (Euphorbiaceae), was revised to be (S)-4,8-dioxo-3-methoxy-2-methyl-5-n-octyl-1,4,5,6,7,8-hexahydroquinoline [(S)-2], not the isoquinoline derivative 1, as assumed previously. The revision was initiated by biosynthetic feeding experiments of one of our groups.
An efficient evaluation procedure for the chemical screening and on-line structural elucidation o... more An efficient evaluation procedure for the chemical screening and on-line structural elucidation of dimeric naphthylisoquinoline alkaloids has been developed. The method is based on the lead tetraacetate oxidation of the central binaphthalene core of the alkaloids. UV spectra of the extracts after addition of the oxidant show, in the presence of naphthylisoquinoline dimers, the appearance of a characteristic long-wavelength absorption indicative of dinaphthoquinones. The efficiency and relevance of the method has been demonstrated in the discovery of a constitutionally and configurationally new dimeric naphthylisoquinoline alkaloid, named ancistrogriffithine A (4), from the previously uninvestigated Asian liana Ancistrocladus griffithii. After verification of this screening result by LC-ESI-MS/MS, the constitution and the relative configuration of the compound were elucidated on line, by LC-NMR and LC-CD on the extract. Using an LC-NMR-WET-ROESY experiment, it was possible for the first time to determine the relative axial configuration of a natural biaryl compound on line, by observing long-range ROE interactions. Finally, an oxidative degradation right on the extract delivered the absolute configuration of 4, without isolation of the alkaloid. Ancistrogriffithine A is the as yet only dimeric naphthylisoquinoline from an Asian Ancistrocladaceae plant.
The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/... more The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/5 Linsmaier and Skoog medium was studied. During a lag phase in growth, droserone concentrations in the medium reached a value of 2.1 mg g À1 fr. wt. After this maximum value the concentration decreased slightly to 1.8 mg g À1 fr. wt., while the growth of the calli was enhanced (25% increase in fr. wt. within 7 days). Plumbagin and isoshinanolone were likewise present in the medium. By feeding 13 C 2-labelled acetate to the cultures the biosynthesis of droserone was elucidated. The incorporation of whole C 2-units unambiguously shows its acetogenic origin and ®ts well in the biosynthetic scheme suggested for the structurally Ð and biogenetically Ð related naphthylisoquinoline alkaloids.
European Food Research and Technology, Apr 30, 2023
The so-called "craft beer revolution" has increased the demand for new styles of beers, often wit... more The so-called "craft beer revolution" has increased the demand for new styles of beers, often with new ingredients like flavour extracts. In recent years, synthetic biology has realized the production of a plethora of plant secondary metabolites in microbial hosts, which could provide an alternative source for these compounds. In this study, we selected a in situ flavour production approach for grape flavour addition. We used an O-methyl anthranilate (OmANT) producing laboratory Saccharomyces cerevisiae strain in co-fermentations with an industrial beer yeast strain WLP644. The laboratory strain provided an ease of genetic manipulation and the desirable properties of the WLP644 strain were not modified in this approach. In shake flasks, a 10:90 ratio of the yeasts produced grape flavoured beer with the yeast produced flavour compound in a range normally used for flavoured beverages. Hopped and unhopped beers were analysed by VTT's trained sensory panel and with olfactory GC-MS. OmANT was successfully detected from the beers as a floral odour and flavour. Moreover, no off-flavours were detected and aroma profiles outside the grape flavour were rather similar. These results indicate that the co-fermentation principle is a suitable approach to change the flavour profiles of beers with a simple yeast strain drop-in approach.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Plants are capable of producing an overwhelming variety of secondary metabolites, both in terms o... more Plants are capable of producing an overwhelming variety of secondary metabolites, both in terms of complexity and quantity. Yet, when compared with the situation in microorganisms this impressive metabolic machinery is hardly exploited. One of the main reasons is the limited molecular insight into plant secondary metabolism, which hampers the design of effective metabolic engineering strategies. To address these shortcomings we employ a functional genomics based technology platform that enables comprehensive investigations and large-scale gene discovery programs in plant metabolism. The platform relies primarily on the integration of genome-wide transcript profiling with metabolite profiling and is applicable to any plant species or system to map the biosynthesis of any metabolite.
In this paper, an analytical method for the analysis of molecular lipids in algae samples is repo... more In this paper, an analytical method for the analysis of molecular lipids in algae samples is reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh performance liquid chromatography combined with mass spectrometry (UPLC-MS). For further characterization of lipids, MS/MS analyses are carried out utilizing either a separate instrument (e.g., LTQ-Orbitrap mass spectrometer) or simultaneous fragmentation with the same instrument. Throughput of the method is over 100 samples/d. The repeatability is good, and the relative standard deviation of spiked samples is <15%.
Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described.... more Constituents of microalgae and sample preparation for UPLC-ELSD and GC-MS analyses are described. Bound fatty acids from acylglycerols, alkylacylglycerols, galactosyldiacylglycerols, glycerophospholipids, and sterol esters are derivatized by using transesterification with sodium methoxide to form fatty acid methyl esters. Compounds containing free hydroxyl groups, either present originally or formed during previous step, like free fatty acids, sterols, α-tocopherol, phytol, and nonesterified alkoxyglycerols, are trimethylsilylated. The compounds in algal lipid extract are subsequently derivatized by these two steps.
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