| Abstract |
In addition to many essential nutritional components, plants contain (poly)phenolic substances that comprise a large and heterogeneous group of biologically active non-nutrient
compounds. Among them flavonoids and stilbenoids are of exceptional interest, with the former consisting of a diverse array of structurally related compounds possessing the same
C6-C3-C6 backbone skeleton, derived from the phenylpropanoid biosynthetic pathway. The term “flavonoids” is used to include the three classes of structurally related compounds
deriving from the condensation reaction of an activated hydroxy-cinnamic acid
(phenylpropanoid) with malonyl-CoA, main flavonoids, isoflavonoids and neoflavonoids. On the other hand stilbenoids are compounds derived by a similar condensation reaction but with
the participation of an evolutionarily diverged enzyme.
The significance of flavonoids and stilbenoids derives from the fact that they are involved in aspects of plant physiology such as tissue pigmentation, defence against microbes or insects, interactions with other organisms, protection against UV irradiation, transmission and response to specific environmental stimuli, pollen germination and active auxin transfer.
Moreover these compounds, as found recently, have also high pharmacological value.
Flavonoids have long been recognised to possess anti-oxidant, anti-inflammatory, antimicrobial,
anti-proliferative, anti-allergenic activities and acting as post-menopausal and cardiovascular cures. Inverse relationships between the intake of flavonoids and the risk of
coronary heart disease, stroke and many types of cancer have been shown by epidemiological studies.
A plethora of secondary metabolites with such exceptional properties are produced through plant’s metabolic machinery in traceable quantities or are extremely difficult to extract or process them. The interest for the exploitation of these metabolite properties by the
pharmaceutical or agricultural industry (biological or integrated crop protection) and medicine
has increased the need for heterologous biosynthesis of substances that are assumed or proven
to possess beneficial actions, such as the flavonoids and stilbenoids. Coumaric acid, a phenolic acid, resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and
flavonols kaempferol and quercetin have been shown to be substances with potential nutritional and agricultural value.
In this doctoral dissertation, six metabolically engineered yeast (Saccharomyces cerevisiae) strains harbouring plasmids with all the necessary genes that permit the
biosynthesis of the abovementioned compounds, utilizing phenylalanine as a precursor, have been constructed. Yeast strains with transcriptionally active heterologous genes, were used to
construct time courses showing the dependence of the precursor utilization and the quantification of the respective end-product synthesis. Thus it has been demonstrated the production of 108,6mg/L of coumaric acid, 0,29-0,31mg/L of resveratrol, 8,9-15,6mg/L of naringenin, 0,1-7,7mg/L of genistein, 0,9-4,6mg/L of kaempferol and 0,26-0,38mg/L of quercetin in culture media, with respect to the precursor molecule that has been tested.
Moreover, experiments were performed to optimize the parameters involved in the heterologous biosynthetic pathways. Especially, the optimal concentration of the precursor
molecule was studied as well as the relevance of starting inoculum at the end-product concentration. Furthermore, the fluxes through the intermediate nodes of the pathways were studied. Finally, the advantages and disadvantages of the use of yeast are discussed as being a host for the heterologous production of flavonoids and stilbenoids and the kind of their
potential uses in wine and agricultural (phytoprotection) industry.
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