Biosynthesis of higher hydroxylated Flavonols

The project Formation of higher hydroxylated flavonols focuses on the establishment of the complex substitution pattern of flavonols, which are important bioactive compounds in plants and in plant derived food. This proposal involves two research teams with specific yet complementary skills and a well-established track record in their respective research fields. The Austrian team has an extensive experience in polyphenol research and has elucidated several steps of the flavonoid pathway in various horticulturally relevant plants. The French team has a solid experience in the development of new synthesis methodologies and in their application to the synthesis of polyphenolic natural products. Flavonols are present in various foods and beverages commonly incorporated in the human diet and are therefore considered as functional dietary ingredients due to their beneficial health effects. In plants, flavonoids fulfil important physiological functions playing crucial roles in e.g. plant growth, pollen fertility, attraction of pollinators and protection against abiotic stress. The substitution pattern of flavonols seems to be of critical importance for the observed physiological effects. Therefore, flavonols with specific structural modification (hydroxylation, methylation, glycosylation) are of special interest. The knowledge on the genesis of higher hydroxylated flavonols, that feature additional hydroxyl groups in position(s) 6 and/or 8 of the A-ring and carry methyl and glycosyl group in various positions, is only partially understood, and the underlying genes are unknown. The aim of this French-Austrian consortiums is to fully elucidate the pathways leading to these higher hydroxylated flavonols by following a combined genomic and chemical proteomic approach aimed at identifying the enzymes responsible for such flavonol modifications and the biochemical factors determining their intervention. Because comon methods do not allow the isolation of the genes of interest, the encoded enzymes will be enriched, isolated and indentified. Thereafter, the obtained protein sequences can be used to isolate the related genes. Flavonol-bearing probes will be created and evaluated for their effectiveness and selectivity towards flavonol converting enzymes using a range of recombinant proteins from the flavonoid pathway. This will particularly include membrane bound proteins, because hydroxylation in position 6 and 8 involves membrane associated enzymes. The studies will use petals of Chrysanthemum segetum to investigate formation of 8-hydroxyflavonols (gossypetin) derivatives and of Tagetes sp. and Rudbeckia hirta for the elucidation of the 6- hydroxyflavonol (quercetagetin) pathway. The genes and proteins will be characterized in detail. The expected results of this project will permit to confirm the value and utility of chemical proteomics using designed polyphenol-bearing probes to address other important yet unsolved questions on polyphenol-protein interactions related to either the biogenesis of polyphenols or the biological activities and impact of dietary plant polyphenols on human health. This will allow to identify for the first time novel genes involved in the formation of higher hydroxylated enzymes. The resulting recombinant enzymes obtained within the project will allow the synthesis or modification of flavonoids to evaluate their potential use as bioactive secondary metabolites or as lead molecules for pharmaceutical development.

Flavonols are present in many foods and beverages and are considered functional dietary ingredients due to their many health benefits. In plants, flavonoids fulfil important physiological functions, playing crucial roles in plant growth, pollen fertility, attraction of pollinators and protection against abiotic stress, for example. The substitution pattern of flavonols seems to be of critical importance for the observed physiological effects. Therefore, flavonols with specific structural modifications (hydroxylation, methylation, glycosylation) are of special interest. The formation of higher hydroxylated flavonols, which feature additional hydroxyl groups in position(s) 6 and/or 8 of the A-ring, and carry methyl and glycosyl groups in various positions, is only partially understood, and the underlying genes are unknown. The project focused on elucidating the complex substitution pattern of flavonols, which are important bioactive compounds in plants and plant derived food. This proposal involved two research teams with specific yet complementary skills and well-established track records in their respective research fields. The Austrian team has extensive experience in polyphenol research and has elucidated several steps of the flavonoid pathway in various horticulturally relevant plants. The French team has solid experience in the development of new synthesis methodologies and in their application to the synthesis of polyphenolic natural products. The French-Austrian consortium followed a combined genomic and chemical proteomic approach, aimed at identifying the enzymes responsible for flavonol modifications. Flavonol-bearing probes were created and evaluated for their effectiveness and selectivity towards flavonol converting enzymes using a range of recombinant proteins from the flavonoid pathway. This specifically included membrane bound proteins, because hydroxylation in positions 6 and 8 involves membrane associated enzymes. The studies used petals of Chrysanthemum segetum to investigate formation of 8-hydroxyflavonols (gossypetin) derivatives and of Tagetes sp. and Rudbeckia hirta for the elucidation of the 6-hydroxyflavonol (quercetagetin) pathway. In parallel, transcriptome analysis of Rudbeckia hirta and Tagetes erecta identified candidate genes for flavonol 6-hydroxylases (F6H) from both plants. Full size cDNA clones were subsequently isolated and expressed recombinantly in S. cerevisiae. The resulting recombinant protein was functionally active and showed the expected F6H activity. The enzymes were characterized. Additionally, gene expression studies of 6-hydroxyflavonol related genes in R. hirta petals were performed. The project allowed the first identification of novel genes involved in the formation of higher hydroxylated enzymes. The recombinant enzymes obtained within the project will enable the synthesis or modification of flavonoids to evaluate their potential use as bioactive secondary metabolites or as lead molecules for pharmaceutical development. The successful project confirmed the value and utility of chemical proteomics using designed polyphenol-bearing probes to address other important yet unsolved questions on polyphenol-protein interactions related to either the biogenesis of polyphenols or the biological activities and impact of dietary plant polyphenols on human health.