Biokatalytische Entstehung von Sesquiterpenoiden

Im Rahmen der vorliegenden Arbeit wurde der Basidiomycet Cerrena unicolor (Cun) als Sesquiterpenproduzent untersucht. Im Genom des Pilzes konnten mit Hilfe von Sequenzvergleichen 14 Sesquiterpensynthasen (STS) identifiziert werden. Zehn von ihnen wurden anschließend erfolgreich heterolog in Escherichia coli exprimiert, die Enzyme produziert und funktionell charakterisiert. Mittels tryptischem Verdau und anschließendem peptide mass fingerprinting konnten die vorhergesagten Proteinsequenzen bestätigt werden. Die Sequenzvergleiche zeigten STS-typische konservierte Motive sowie möglicherweise für Cun spezifische Homologien. Für die Untersuchung der Metallionenabhängigkeit wurden zwei STS aus Cun gewählt, die sich sowohl in der Anzahl der Produkte, als auch den produzierten Sesquiterpenen selbst unterschieden. Setzte man andere bivalente Metallionen als Mg2+ für die Zyklisierungsreaktion ein, zeigte sich, dass die ausgewählte Multiprodukt-Cyclase promiskuitiver als die Cyclase mit nur einem Sesquiterpen als Produkt reagierte. Weiterhin konnte gezeigt werden, dass agro-industrielle Nebenströme für die Kultivierung von Cun verwendet werden können und diese Einfluss auf die Sesquiterpenbiosynthese hatten. Eher kohlenhydratreiche Nebenströme wie Kartoffelstärke oder Weizenkleie hatten keinen positiven Einfluss auf die Produktausbeute. Für den lipidhaltigen Rapspresskuchen konnte jedoch eine deutliche Erhöhung der Produktausbeute nachgewiesen werden. Die Hypothese, dass durch β-Oxidation der Lipide ein Überfluss an Acetyl-CoA die Sesquiterpenproduktion boostet, konnte mittels 13C-markiertem Acetat als Medienzusatz experimentell bestätigt werden.Aroma compounds play a key role in the food industry, with customers paying increasing attention to sustainability and naturalness. In order to achieve this, focus was put on higher fungi as a source of aroma compounds and enzymes for production and quality improvements of foods. In this study, Cerrena unicolor (Cun) was investigated as a producer of sesquiterpenes. Using sequence alignments, 14 sesquiterpene synthases (STS) were identified in the fungal genome. Ten of them were successfully expressed in Escherichia coli, the enzymes produced and functionally characterized. Using tryptic digestion followed by peptide mass fingerprinting, the predicted protein sequences were confirmed. Sequence alignments revealed STS-type conserved motifs, as well as homologies possibly specific for Cun. To investigate metal ion dependence, two STS were selected, which differed in the total number of products and in the products it self. Using other divalent metal ions than Mg2+ for the cyclization reaction showed that the chosen multiproduct cyclase was more promiscuous than the cyclase producing only one single sesquiterpene. Furthermore, it was shown that agro-industrial side streams can be used for the cultivation of Cun and influence sesquiterpene biosynthesis. Carbohydrate-containing side streams such as potato starch and wheat bran did not have a positive influence on the product yield. However, a significant increase in product yield was demonstrated for the lipid-containing rapeseed press cake. An excess of acetyl-CoA caused by the β-oxidation of respective lipids boosted sesquiterpene production, which was confirmed by the incorporation of 13C-labeled acetate supplemented to the nutrient medium

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool

Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor

Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity