Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz , thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol ( 1 ) and its ketone lup-19(21)-en-3-one ( 2 ). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, β-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

The Russian dandelion Taraxacum koksaghyz produces high-value isoprenoids such as pentacyclic triterpenes and natural rubber in the latex of specialized cells known as

In the past, the topic of excessive salt consumption associated with various chronic diseases became a focus of nutritional science. Enzymes such as hydrolases and specifically peptidases are widely used in industry since they do not require cofactors, are often stable at high pH values, and have a broad cleavage specificity. Thus, a new approach to use peptidases would be the generation of L‐arginyl dipeptides, which do not have a taste on their own, but are able to enhance the salty taste. This thesis should focus on the isolation and characterisation of arginyl‐specific peptidase genes from basidiomycetes to generate these dipeptides.First, various peptidase genes from Trametes versicolor, Phanerochaete chrysosporium and Schizophyllum commune were isolated and expressed in Escherichia coli or Komagataella phaffii. The serine (ABB73029) peptidase from P. chrysosporium and the aspartate peptidase from T. versicolor (EIW62808) were successfully purified. Activity assays showed that the aspartate peptidase from T. versicolor is not an arginyl‐specific peptidase, while the analysis of cleavage sites indicates that the serine peptidase from P. chrysosporium is an exopeptidase. Next, 29 basidiomycetes were cultivated over 24 days in minimal medium with 1 % gluten and analysed regarding their endo‐ and exopeptidase activity. Out of 29 fungi, five interesting species with arginyl‐specific peptidase activity were chosen, namely Agrocybe aegerita, Fomitop‐ sis pinicola, Flammulina velutipes, Hypholoma sublateritium and Pleurotus eryngii. After purification of the culture supernatant using size exclusion chromatography, active fractions were used to hydrolyse the substrate casein, and cleavage products were analysed. Cleavage sites were only detected for A. aegerita, F. velutipes and P. eryngii. Beside arginyl‐specific cleavage sites also a high amount of undesired exopeptidase cleavage sites was determined assuming that these peptidases are probably exopeptidases rather than arginyl‐specific peptidases.Generation of salt taste enhancing dipeptides should finally be achieved with a dipeptidyl‐ peptidase V (DPPV) from Pleurotusfloridanus. PflDPPV was heterologously produced in E. coli, had optima at pH 8.5 and 60 °C and was not completely inhibited by the tested peptidase inhibitors.