Biochemical Genetics of Metabolic Plasticity

Bitter taste perception cautions humans against the ingestion of potentially toxic compounds. However, current knowledge about natural bitter substances and their activation of human bitter taste receptors (TAS2Rs) is biased toward substances from flowering plants, whereas other sources are underrepresented. Although numerous mushrooms taste bitter, the corresponding substances and receptors are unexplored. Three previously undescribed triterpene glucosides, named oligoporins D−F, together with the known oligoporins A and B, were isolated from Amaropostia stiptica. The structures of oligoporins D−F were determined using spectroscopic analyses. The isolated oligoporins and the bitter indolalkaloid infractopicrin from Cortinarius infractus were functionally screened with all TAS2Rs. For all compounds, at least one responding receptor was identified. Oligoporin D activated TAS2R46 already at a submicromolar concentration and thus belongs to the family of most potent bitter agonists. The addition of mushroom compounds to the list of cognate TAS2R activators lowers the existing bias of knowledge about bitter agonists.

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

Seeds of domesticated Vicia (vetch) species (family Fabaceae-Faboideae) are produced and consumed worldwide for their nutritional value. Seed accessions belonging to 16 different species of Vicia—both domesticated and wild taxa—were subjected to a chemotaxonomic study using ultraperformance liquid chromatography–mass spectrometry (UPLC-MS) analyzed by chemometrics. A total of 89 metabolites were observed in the examined Vicia accessions. Seventy-eight out of the 89 detected metabolites were annotated. Metabolites quantified belonged to several classes, viz., flavonoids, procyanidins, prodelphinidins, anthocyanins, stilbenes, dihydrochalcones, phenolic acids, coumarins, alkaloids, jasmonates, fatty acids, terpenoids, and cyanogenics, with flavonoids and fatty acids amounting to the major classes. Flavonoids, fatty acids, and anthocyanins showed up as potential chemotaxonomic markers in Vicia species discrimination. Fatty acids were more enriched in Vicia faba specimens, while the abundance of flavonoids was the highest in Vicia parviflora. Anthocyanins allowed for discrimination between Vicia hirsuta and Vicia sepium. To the best of our knowledge, this is the first report on employing UPLC-MS metabolomics to discern the diversity of metabolites at the intrageneric level among Vicia species.

Late blight, caused by the oomycete Phytophthora infestans, is economically the most important foliar disease of potato. To assess the importance of the leaf surface, as the site of the first encounter of pathogen and host, we performed untargeted profiling by liquid chromatography–mass spectrometry of leaf surface metabolites of the susceptible cultivated potato Solanum tuberosum and the resistant wild potato species Solanum bulbocastanum. Hydroxycinnamic acid amides, typical phytoalexins of potato, were abundant on the surface of S. tuberosum, but not on S. bulbocastanum. One of the metabolites accumulating on the surface of the wild potato was identified as lysophosphatidylcholine carrying heptadecenoic acid, LPC17:1. In vitro assays revealed that both spore germination and mycelial growth of P. infestans were efficiently inhibited by LPC17:1, suggesting that leaf surface metabolites from wild potato species could contribute to early defense responses against P. infestans.

Rosemary and sage species from Lamiaceae contain high amounts of structurally related but diverse abietane diterpenes. A number of substances from this compound family have potential pharmacological activities and are used in the food and cosmetic industry. This has raised interest in their biosynthesis. Investigations in Rosmarinus officinalis and some sage species have uncovered two main groups of cytochrome P450 oxygenases that are involved in the oxidation of the precursor abietatriene. CYP76AHs produce ferruginol and 11-hydroxyferruginol, while CYP76AKs catalyze oxidations at the C20 position. Using a modular Golden-Gate-compatible assembly system for yeast expression, these enzymes were systematically tested either alone or in combination. A total of 14 abietane diterpenes could be detected, 8 of which have not been reported thus far. We demonstrate here that yeast is a valid system for engineering and reconstituting the abietane diterpene network, allowing for the discovery of novel compounds with potential bioactivity.

Lens culinaris and several Lupinus species are two legumes regarded as potential protein resources aside from their richness in phytochemicals. Consequently, characterization of their metabolite composition seems warranted to be considered as a sustainable commercial functional food. This study presents a discriminatory holistic approach for metabolite profiling in accessions of four lentil cultivars and four Lupinus species via gas chromatography/mass spectrometry. A total of 107 metabolites were identified, encompassing organic and amino acids, sugars, and sterols, along with antinutrients, viz., alkaloids and sugar phosphates. Among the examined specimens, four nutritionally valuable accessions ought to be prioritized for future breeding to include Lupinus hispanicus, enriched in organic (ca. 11.7%) and amino acids (ca. 5%), and Lupinus angustifolius, rich in sucrose (ca. 40%), along with two dark-colored lentil cultivars ‘verte du Puy’ and ‘Black Beluga’ enriched in peptides. Antinutrient chemicals were observed in Lupinus polyphyllus, owing to its high alkaloid content. Several species-specific markers were also revealed using multivariate data analyses.

Ceramides (Cers) are major components of the outermost layer of the skin, the stratum corneum, and play a crucial role in permeability barrier functions. Alterations in Cer composition causing skin diseases are compensated with semisynthetic skin-identical Cers. Plants constitute new resources for Cer production as they contain glucosylceramides (GluCers) as major components. GluCers were purified from industrial waste plant materials, apple pomace (Malus domestica), wheat germs (Triticum sp.), and coffee grounds (Coffea sp.), with GluCer contents of 28.9 mg, 33.7 mg, and 4.4 mg per 100 g of plant material. Forty-five species of GluCers (1–45) were identified with different sphingoid bases, saturated or monounsaturated α-hydroxy fatty acids (C15–28), and β-glucose as polar headgroup. Three main GluCers were hydrolyzed by a recombinant human glucocerebrosidase to produce phyto-Cers (46–48). These studies showed that rare and expensive phyto-Cers can be obtained from industrial food plant residues.

Ceramides (Cers) are major components of the outermost layer of the skin, the stratum corneum, and play a crucial role in permeability barrier functions. Alterations in Cer composition causing skin diseases are compensated with semisynthetic skin-identical Cers. Plants constitute new resources for Cer production as they contain glucosylceramides (GluCers) as major components. GluCers were purified from industrial waste plant materials, apple pomace (Malus domestica), wheat germs (Triticum sp.), and coffee grounds (Coffea sp.), with GluCer contents of 28.9 mg, 33.7 mg, and 4.4 mg per 100 g of plant material. Forty-five species of GluCers (1–45) were identified with different sphingoid bases, saturated or monounsaturated α-hydroxy fatty acids (C15–28), and β-glucose as polar headgroup. Three main GluCers were hydrolyzed by a recombinant human glucocerebrosidase to produce phyto-Cers (46–48). These studies showed that rare and expensive phyto-Cers can be obtained from industrial food plant residues.

This study aimed to evaluate the influence of different light regimens during spinach cultivation on the isomeric composition of β-carotene. Irradiation with a halogen lamp, which has a wavelength spectrum close to that of daylight, was used to mimic field-grown conditions. The additional use of optical filters was established as a model system for greenhouse cultivation. Field-grown model systems led to a preferential increase of 9-cis-β-carotene, whereas 13-cis-β-carotene was just formed at the beginning of irradiation. Additionally 9,13-di-cis-β-carotene decreased significantly in the presence of energy-rich light. Isomerization of β-carotene was strongly suppressed during irradiation in greenhouse-grown model systems and led to significant differences. These results were verified in biological samples. Authentic field-grown spinach (Spinacia oleracea L.) showed among changes of other isomers a significantly higher level of 9-cis-isomers (7.52 ± 0.14%) and a significantly lower level of 9,13-di-cis-isomers (0.25 ± 0.03%) compared to authentic greenhouse-grown spinach (6.49 ± 0.11 and 0.76 ± 0.05%). Almost all analyzed commercial spinach samples (fresh and frozen) were identified as common field-grown cultivation. Further investigations resulted in a clear differentiation of frozen commercial samples from fresh spinach, caused by significantly higher levels of 13-cis- and 15-cis-β-carotene as a result of industrial blanching processes.

This study aimed to evaluate the influence of different light regimens during spinach cultivation on the isomeric composition of β-carotene. Irradiation with a halogen lamp, which has a wavelength spectrum close to that of daylight, was used to mimic field-grown conditions. The additional use of optical filters was established as a model system for greenhouse cultivation. Field-grown model systems led to a preferential increase of 9-cis-β-carotene, whereas 13-cis-β-carotene was just formed at the beginning of irradiation. Additionally 9,13-di-cis-β-carotene decreased significantly in the presence of energy-rich light. Isomerization of β-carotene was strongly suppressed during irradiation in greenhouse-grown model systems and led to significant differences. These results were verified in biological samples. Authentic field-grown spinach (Spinacia oleracea L.) showed among changes of other isomers a significantly higher level of 9-cis-isomers (7.52 ± 0.14%) and a significantly lower level of 9,13-di-cis-isomers (0.25 ± 0.03%) compared to authentic greenhouse-grown spinach (6.49 ± 0.11 and 0.76 ± 0.05%). Almost all analyzed commercial spinach samples (fresh and frozen) were identified as common field-grown cultivation. Further investigations resulted in a clear differentiation of frozen commercial samples from fresh spinach, caused by significantly higher levels of 13-cis- and 15-cis-β-carotene as a result of industrial blanching processes.