Natural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards... Show moreNatural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards this end, using microbiological methods, fungal endophytes were isolated and identified, by morphological characters and PCR-based methods, from vanilla pods, the site of vanilla flavor. The fungal endophytes were tested for their involvement in the synthesis of vanilla flavor. In this way, three vanilla pod endophytes Hypoxylon investiens, Pestalotiopsis microspora, Diaporthe phaseolorum were found to modify the amounts of vanilla flavor and aroma metabolites vanillin, vanillyl alcohol, p-hydroxybenzoic acid, p-coumaric acid, p-xylene, α-phellandrene, 3-carene, α-terpineol, p-hydroxybenzaldehyde, α-cubebene, β-caryophyllene in Vanilla material containing media (in vitro) as well as in living Vanilla plant material (in vivo - acclimatized plants and calli). These metabolites were synthesized either de novo or through biotransformation reactions of precursors. That the latter describes the involvement of several metabolites related to vanilla flavor, implies complex flavor notes, typical of natural vanilla flavor, as opposed to the synthetic version. The analytical methods used include GC-MS, HPLC, 1H and 2D NMR. The results support the hypothesis that endophytes play a role in vanilla flavor metabolite biosynthesis. 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
