Actinobacteria are well known for the production of bioactive natural products, many of which have applications in the fields of human, animal and plant health. Subject of this thesis are the... Show moreActinobacteria are well known for the production of bioactive natural products, many of which have applications in the fields of human, animal and plant health. Subject of this thesis are the anthracyclines, glycosylated aromatic polyketides with potent anticancer activity. Despite remarkable efficacy against acute leukaemia and various solid tumours, their application is limited by severe side effects, such as cardiotoxicity, therapy-related tumours and infertility.Recent insights into the mode-of-action of anthracyclines have prompted renewed interest into this important class of anticancer compounds. One of the most promising compounds is N,N-dimethyldoxorubicin, which exerts significantly reduced side effects. To date, DMdoxo has not been isolated from natural sources, but biosynthesis would be an attractive option for scaling up production of this promising compound.For this reason, we aimed to engineer the Doxo biosynthetic pathway in S. peucetius for the production of biosynthetic N,N-dimethylated anthracyclines. The challenges that we encountered instigated the study of anthracycline resistance mechanisms in Streptomyces, and potential applications of detoxified anthracyclines as antibiotics. Finally, to facilitate future screening and strain engineering efforts of Streptomyces producer strains, we developed a workflow for integrated quantitative proteomics and metabolomics from small-scale Streptomyces cultures Show less
Mohd Zuwairi Bin Saiman, M.; Mustafa, N.R.; Choi, Y.H.; Verpoorte, R.; Schulte, A.E. 2015
The research described in this thesis was aimed at analyzing the regulation of the channeling of carbon into different terpenoid pathways including the terpenoid indole alkaloids (TIA) and their... Show moreThe research described in this thesis was aimed at analyzing the regulation of the channeling of carbon into different terpenoid pathways including the terpenoid indole alkaloids (TIA) and their iridoid precursors in Catharanthus roseus cell suspension cultures. Of the different terpenoid classes; monoterpenoid (C10; TIA), triterpenoid (C30; sterol), and tetraterpenoid (C40; carotenoid), the latter was the largest pool. The production of carotenoids and TIA through the methyl-erythritol phosphate (MEP) pathway was increased after jasmonic acid elicitation, while the sterols from the mevalonate (MVA) pathway were not increased. Mevalonate feeding only increased the sterols. The effect of geraniol feeding alone and in combination with jasmonic acid elicitation resulted in increased levels of loganic acid and strictosidine, respectively. Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastid or cytosol were successfully developed and metabolic analysis by NMR-based metabolomics showed different metabolic effects related to the subcellular compartmentation of geraniol synthase. In conclusion, reallocation of C5 units between MEP and MVA pathway seems difficult, but both have different regulation which could be further studied to increase alkaloid production. Show less
The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological... Show moreThe (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anti-cancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes in an alternative plant host allowed the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programs towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications. Show less
