Characterisation of two novel oleaginous yeast strains for efficient and sustainable production of acetyl-CoA derived products

Abstract: Yeasts are major players in industry for production of valuable compounds. The market for yeast bioproduction is constantly growing, and sustainable bioproduction from renewable feedstocks is becoming increasingly important. Previously, Sacccharomyces cerevisiae has been the key player in research and industrial applications, and engineered strains have been created to have many features already present in other yeast species (production of valuable products, and utilisation of pentose sugars as carbon sources, etc.). The efficient use of pentoses is a highly investigated topic due to their abundance in renewable feedstocks such as lignocellulosic hydrolysates. Numerous yeast species exist that are naturally capable of assimilating pentose sugars with greater efficiency than engineered Saccharomyces .  These species can also produce industrially relevant products like terpenoids, enzymes, and bioactive substances. Using the biodiversity naturally available among yeasts is an important step towards the improvement of sustainable bioproduction.   This thesis focuses on and discusses the importance of non-conventional yeasts, their isolation, characterization, and potential bioproduction of acetyl-CoA derived compounds. Two novel, non-conventional basidiomycete oleaginous yeast strains were investigated in detail: BOT-O, a Pseudozyma hubeiensis strain with high levels of storage lipid and biosurfactant production, and BOT-A2, a Rhodotorula toruloides strain, which produces of high-value carotenoids and storage lipids. Both were isolated from plant material and characterised regarding physiology, genomics and transcriptomics. The strains were de novo sequenced, their genomes assembled and annotated. RNA sequencing was conducted to investigate the differences in transcriptional levels between growth on xylose and glucose, or during nitrogen starvation, which is an important condition for high lipid production. Both BOT-O and BOT-A2 produced high amounts of lipids on glucose or xylose. BOT-O showed identical growth rates on both sugars. Both were also somewhat tolerant towards lignocellulosic inhibitors. Interestingly, BOT-O was more tolerant towards furanic aldehydes, while BOT-A2 was more tolerant towards weak acids. Six new promoters that are regulated during nitrogen starvation were identified for BOT-A2. They are a valuable addition to the R. toruloides genetic engineering toolbox. Overall, these two new isolates have highly interesting characteristics and the potential to become hosts for bioproduction of native and non-native acetyl-CoA derived compounds.