Establishing the production of polyprenylated xanthones in engineered yeast

Over centuries, Hypericum species like Hypericum perforatum have been utilized all around the world due to their medicinal properties, attracting significant attention from research groups in both scientific and industrial domains globally. Throughout the time, extensive efforts were made for the identification, analysation and evaluation of the primary substrates naturally synthesized by these genus members, responsible for their interesting pharmacological properties. These efforts resulted in the discovery of various bioactive compounds. Among these compounds, hyperixanthone A stands out as a notable example, belonging to the polyprenylated xanthones, this substrate showed potent antibacterial activity against multidrug-resistant Staphylococcus aureus. Exhibiting this highly interesting biological activity the extraction of hyperixanthone A, but also various other polyprenylated xanthones, showing pharmacological relevant biological activities, was attempted. While being successful these extractions were time consuming and also resulted in an overall low yield of the extracted substrate. Consequently, efforts have been directed towards the elucidation of the biosynthetic pathway of hyperixanthone A across different Hypericum species, resulting in the total discovery of its biosynthesis pathway in plants with just the final reverse prenylation step remaining elusive up to this day. With the sequences of all necessary enzymes catalysing each step of the biosynthesis pathway of hyperixanthone A, up to the last intermediate patulone, known and their activity tested and analysed during multiple in vitro activity assays, this study focused at the in vivo establishing of the biosynthesis pathway from 2,4,6-trihydroxybenzophenone to patulone, in engineered yeast, aiming for an overall enhanced substrate extraction efficiency and substrate yield. Navigating through multiple challenges associated with the in vivo production of patulone regarding each of the five involved enzymes, a variety of optimization strategies were analysed and tested. These optimisation approaches reached from expression system switches and modifications, sequence truncations, the development and optimisation of a new cultivation method and the evaluation of diverse yeast strains to the localization and expression level analysis of specific enzymes via confocal fluorescence microscopy. All of these optimisation attempts contributed to their own extents to the final achievement of the first ever detected in vivo production of patulone in engineered yeast. This achievement not only demonstrated a more efficient way for a later on production for hyperixanthone A but also showcased a wide variety of optimisation approaches and their enhancing effects on the in vivo production of polyprenylated xanthones like patulone in engineered yeast, paving the way for yeast-based production platforms capable of supplying these compounds for further scientific research and industrial applications.