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Flavonolignan Biosynthesis in Silybum marianum – Potential Regulatory Mechanisms and Candidate Genes
Silymarin, a flavonolignan mixture from milk thistle (Silybum marianum, Asteraceae), is mainly used for the supportive therapy of chronic liver diseases or to prevent toxic liver damage. In addition, beneficial effects for human health like tumor inhibition and immunomodulatory mechanisms have been...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2017
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| Zusammenfassung: | Silymarin, a flavonolignan mixture from milk thistle (Silybum marianum, Asteraceae), is mainly used for the supportive therapy of chronic liver diseases or to prevent toxic liver damage. In addition, beneficial effects for human health like tumor inhibition and immunomodulatory mechanisms have been reported. The plant produces and accumulates silymarin mainly in the testa and pericarp for protection of its sporophytic embryo against plant herbivores and to limit or prevent bacterial, fungal or viral infections.
The final step in the biosynthetic pathway of silymarin is not yet fully elucidated. Starting point is the amino acid L-phenylalanine which is converted to 4-coumaroyl-CoA which is the essential precursor for flavonoids and monolignols. A flavonoid (taxifolin) and a monolignol (coniferyl alcohol) then are transformed to silymarin by radical formation (oxidation), coupling, rearrangement and subsequent cyclisation. Several positional isomers (mainly silychristin, silydianin, silybin and isosilybin) occur, the latter two being present as diastereomeric pairs. The distribution of these regioisomers differs in ecotypes/cultivars and genotypes. Whereas a peroxidase (POD) is probably responsible for the radical formation, very little is known about how the plant discriminates between its regioisomers and diastereomers. A potential involvement of dirigent proteins (DIRs), controlling the formation, has been investigated.
In vitro cultures have been established from seedlings of three Silybum marianum varieties for further insights into flavonolignan content and composition in suspension cultures and the release of flavonolignans to the outer compartments. Additionally, over a period of two weeks, one culture line was characterised based on various medium parameters, silymarin formation and corresponding enzyme activities. The same culture line was also subjected to an elicitation attempt. However, despite a slight increase in the quantities of the specialised metabolites of the silymarin mixture, the amounts of flavonolignans produced in suspension cells of Silybum marianum are very low.
Interestingly, a connection between the individual positional isomer amounts of silymarin extracted from the mature fruit skins and the respective in vitro cells could be determined. The regioisomer composition in suspension cells resembled the chemotype of the plant origin. This underlined the assumption of the existence of high genotypic variations and the possible presence of involved regulatory mechanisms. Higher taxifolin concentrations increased the formation of two specific regioisomers in enzyme assays but no further direct mechanism for discrimination could be found. Since enzyme activity and flavonolignans could also be detected in the medium of suspension cells, transport systems might play a role as well.
A class III secretory peroxidase, a laccase of the cupredoxin superfamily and two different dirigent proteins could be identified to be a part of the gene pool of Silybum marianum. Unfortunately, the expression of these genes in different E. coli and yeast strains turned out to be a big hurdle. Signal peptides and several glycosylation sites, crucial for structure and activity, greatly contribute to this. Optimisation at some points would be necessary in order to yield recombinant proteins.
On the other hand, the enzyme(s) involved in the coupling reaction between taxifolin and coniferyl alcohol could be extracted from in vitro cells and the respective medium. The radical-forming protein could successfully be identified as a peroxidase with a molecular weight of about 45 kDa using chromatographical methods. In general, structure and function should be similar to the versatile used horseradish peroxidase (HRP). However, this protein alone could not specifically regulate the formation of individual silymarin regioisomers. Even though dirigent proteins could be identified on the genomic level their presence in enzyme preparations or an actual involvement could never be proven. Their expression and utilisation could be plant organ- and/or time-specific, namely active only during the fruit development and maturation phase.
In summary, in the scope of this thesis, enzymes probably involved in the final step of the silymarin biosynthesis and numerous factors possibly regulating the positional isomer ratios were discussed and highlighted. While issues concerning the expression of recombinant proteins remained to be challenging, reasonable solutions were presented. A continuation of this project seems promising and very interesting for further clarification of this subject. |
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| Umfang: | 179 Seiten |
| DOI: | 10.17192/z2017.0674 |