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
Plants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features... Show morePlants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features of SMs are their striking chemical diversity and inter- or intraspecific variation. Due to the large number, high structural diversity and multifunctionality of SMs, it is still an ongoing challenge to understand how this SM diversity comes about, and why such a large diversity is maintained in nature. In this thesis this question was studied using the pyrrolizidine alkaloids (PAs) of Jacobaea species as the study system from an evolutionary and biosynthetic perspective. PA variations were studied among and within Jacobaea species, and species-specific PA profiles were observed. In order to understand how PA diversity is related to species phylogeny, the evolutionary histories and phylogenetic signals of individual PAs were investigated under the phylogenetic context of Jacobaea species and no strong phylogenetic signals were found. To shed light on the mechanisms underlying PA diversity, a gene-to-metabolite approach targeting cytochrome P450 monooxygenases which play an important role in the evolution of chemical diversity was applied to study their involvement in PA biosynthesis and PA diversity. Show less
The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation... Show more The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation sequencing revealed that the biosynthetic potential of actinomycetes had been grossly underestimated. In this thesis, different antibiotics-eliciting strategies, including microbial cocultivation, streptomycin-resistant mutation, overexpression of pathway-specific activator, variation of culture conditions, were utilized to enforce fluctuations in the production of bioactive compounds in actinomycetes, after which, NMR-based metabolic profiling was used to facilitate uncovering those elicited molecules. This pipeline allowed the discovery of new antibiotics involving various chemical skeletons, such as 7-prenylisatin, methoxylated isocoumarins, endophenazines, and C-glycosylpyranonaphthoquinones. On the other hand, genome-mining methodology enabled the discovery of a group of endophenasides and leucanicidin in Kitasatospora sp. MBT66, whereby the rhamnosylation of both scaffold are executed by a same promiscuous glycosyltransferase. Last but not least, a novel antibiotic termed lugdunomycin with unprecedented chemical scaffold, as well as a number of new angucycline-type antibiotics, were characterized from Streptomyces sp. QL37. The biosynthetic pathway of lugdunomycin was deciphered by genetic knockout and OSMAC (One Strain MAny Compound) strategy. In summary, this thesis explores an interface of genomics and metabolomics to accelerate new antibiotics discovery. 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
Salicylic acid (SA) is an important signal compound in systemic acquired resistance in plants. The level of this C6C1 compound in plants increases after a pathogenic attack. There are two... Show moreSalicylic acid (SA) is an important signal compound in systemic acquired resistance in plants. The level of this C6C1 compound in plants increases after a pathogenic attack. There are two biosynthetic pathways of SA, the phenylalanine pathway, which is thought to occur in plants, and the isochorismate pathway, which is found in microorganisms. The biosynthetic pathway of SA in plant is still a matter of debate and some studies showed that the presence of the microbial pathway in plants is also plausible. The present study deals with the production of SA in Catharanthus roseus cell suspension cultures after elicitation with a fungal elicitor (Pythium aphanidermatum extract), the development of a purification method for salicylic acid from plant extract using an ion exchange chromatography, 13C-NMR analysis of purified SA-containing extracts of Catharanthus roseus cells after a labeling experiment with [1-13C]glucose and metabolic profiling of Catharanthus roseus cells elicited with SA. Catharanthus roseus is an important medicinal plant for the production of the anti-tumor agents vincristine and vinblastine, and an excellent model for a retrobiosynthetic study as shown in some studies. We showed by chemistry that in Catharanthus roseus cell suspension cultures, SA is synthesized via the isochorismate pathway. Show less
