Production of carotenoids and lipids by Rhodotorula toruloides CBS 14 cultivated on wheat straw hydrolysate was investigated. An ultra-high-performance liquid chromatography (UHPLC) method for carotenoid quantification was developed and validated. Saponification effects on individual carotenoid quantification were identified, and lipid and carotenoid kinetics during cultivation were determined. The carotenoids β-carotene, γ-carotene, torularhodin, and torulene were identified; β-carotene was the major carotenoid, reaching a maximum of 1.48 mg/100 g dry weight. Recoveries of the carotenoids were between 66 and 76%, except torulene and torularhodin, which had lower recoveries due to saponification effects. Total carotenoid content in saponified and unsaponified yeast extract, respectively, determined by UHPLC or photometer, respectively, was 1.99 mg/100 g and 4.02 mg β-EQ/100 g dry weight. Growth kinetics showed a positive correlation between carotenoid content and lipid accumulation. β-carotene was the major carotenoid at all time points. At the end of the cultivation, triacylglycerols (TAGs) were the major lipid class, with 58.1 ± 3.32% of total lipids. There was also a high proportion of free fatty acids, reaching from 20.5 to 41.8% of total lipids. Oleic acid (C18:1) was the major fatty acid. The lipid yield at the end of the cultivation was 0.13 g/g of sugar consumed.

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The enzymatic hydrolysis of lignocellulosic material in nature is carried out by a plethora of cellulases. Glycoside hydrolase family 45 (GH45) enzymes are small cellulases most commonly found in fungi that catalyse the hydrolysis of β(1→4) linked glucans. Additionally, GH45 enzymes display a structural resemblance to non-hydrolytic protein groups such as expansins and loosenins. In this thesis, the distinctness of GH45 enzymes from different subfamilies was explored. The enzymatic activity of GH45 enzymes from Humicola insolens (HiCel45A), Mytilus edulis (MeCel45A), Trichoderma reesei (TrCel45A), Phanerochaete chrysosporium (PcCel45A), Gloeophyllum trabeum (GtCel45A) was demonstrated. Among the tested substrates were barley betaglucan, konjac glucomannan, carboxymethyl cellulose, and cellohexaose. Initial hydrolysis rates and hydrolysis yields were determined by reducing sugar assays, product formation was analysed using NMR spectroscopy and HPLC. The subfamily B and C enzymes exhibited mannanase activity, and the subfamily B enzyme MeCel45A appeared cold adapted in comparison to TrCel45A. GH45 enzymes are known to act using an inverting action mechanism. This action mechanism had not been experimentally demonstrated in subfamilies B and C, however. Here experimental evidence is provided for the inverting nature of GH45 enzymes from all subfamilies. Comparisons of GH45 enzyme structures were carried out and the first crystal structure of a GH45 subfamily C enzyme from the brown-rot fungus G. trabeum was reported at 1.3 Å resolution. Furthermore, structure and function investigations were done on an isotopically labelled Cel45A from the white-rot fungus P. chrysosporium using NMR spectroscopy. PcCel45A was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1 H, 13C and 15N backbone resonances was obtained and the interaction of 15Nlabelled PcCel45A with cellobiose was studied.