Investigations on the formation of rosmarinic acid in Anthoceros agrestis and Melissa officinalis.
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Philipps-Universität Marburg
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Abstract
Rosmarinic acid (RA) is a natural product found in Anthoceros agrestis with amounts of up to
4 % of dry weight investigated in this work. The biosynthetic pathway for RA has been fully
elucidated for Plectranthus scutellarioides with L-tyrosine and L-phenylalanine serving as
precursors. An essential step in this biosynthesis is the ester linkage of 4-coumaroyl-CoA and
4-hydroxyphenyllactate by rosmarinic acid synthase (RAS) to form
4-coumaroyl-4’-hydroxyphenyllactate which is then further hydroxylated to form RA. However,
for A. agrestis no ortholog to RAS has been found. With RAS being an acyltransferase of the
BAHD family a search for sequences containing conserved motifs characteristic for BAHD
acyltransferases resulted in several sequences, with AaHCT6 being the most similar to RAS
from P. scutellarioides. However, investigations on heterologously expressed AaHCT6 showed,
that it does not catalyze the reaction of 4-coumaroyl-CoA and 4-hydroxyphenyllactate or
3,4-dihydroxyphenyllactate, as it was initially proposed (Petersen et al. 1993; Petersen 1997;
Petersen and Simmonds 2002). A catalogue of 55 potential acceptor substrates was tested for
acceptance by AaHCT6 and resulted in acceptance for shikimate, 3-hydroxyanthranilate and
2,3-dihydroxybenzoate with caffeoyl-CoA and 4-coumaroyl-CoA as donor substrates. Enzyme
kinetic measurements revealed the highest affinity for 4-coumaroyl-CoA as donor substrate
and shikimate as acceptor substrate, followed by 3-hydroxyanthranilate and 2,3-
dihydroxybenzoate. For 3-hydroxyanthranilate the product formation occurs via amide linkage,
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whereas with shikimate and 2,3-dihydroxybenzoate product formation occurs via ester
linkage.
AaHCT1 was successfully amplified from total RNA, ligated into several expression vectors and
introduced into prokaryotic and eukaryotic organisms for heterologous expression. However,
an expression of the protein of interest was not successful. AaHCT2 was successfully expressed
in E. coli and investigations on substrate acceptance revealed an acceptance for two small
alkanols as acceptor substrates, methanol and ethanol, with caffeoyl-CoA as donor substrates.
AaHCT5 was successfully expressed in E. coli as verified by Western blot. However, no substrate
accepted by this enzyme was found out of the catalogue of 55 potential substrates. The
sequence coding for AaHCT7, although discovered in a late stage of this work, was successfully
amplified from total RNA. An incorporation into the vector pDrive was successful as verified by
sequencing. However, an incorporation into the expression vector pET-15b was not successful
as it was implemented in a reverse orientation as verified by sequencing. To date, the ester-
forming step of the rosmarinic acid pathway for Anthoceros agrestis is still unclear. Chances
are that this step is being catalyzed by an enzyme of the BAHD acyltransferase family, as it is
demonstrated for plants of the Lamiaceae family. However, it cannot be ruled out that this
step might be catalyzed by an enzyme of a completely different class. Furthermore, it is
possible that this step might be catalyzed by an enzyme exhibiting acyltransferase activity but
lacking (or deviating from) the conserved sequences typical for BAHD acyltransferases.
In addition to elucidating the function and role of HCTs found in A. agrestis, this work also
delves into the exploration of electricity as a potential elicitor for the production of the
specialized metabolite rosmarinic acid in suspension cultures of Melissa officinalis. Initial
experiments involving electric fields demonstrated varying effects on metabolite
accumulation, indicating the need for further optimization and investigation into the
application of electric current as an elicitor. However, the results showed that the application
of a chemical elicitor, methyl jasmonate, consistently outperforms electric stimulation in
enhancing RA production, highlighting the economic practicality of methyl jasmonate as a
preferred elicitor for specialized metabolite enhancement rather than complex setups with
electric wiring.
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Dates
Created: 2024Issued: 2024-12-09Updated: 2024-12-09
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
BAHD acyltransferasesBAHD-AcyltransferasenBiochemiephenylpropanoid pathwayEnzymologyenzymologybiochemistrymolecular biologyMolekularbiologiePharmaceutical biologyPhenylpropanoidwegPharmazeutische Biologie
DFG-subjects
RosmarinsäureRosmarinsäure SynthaseMelissa officinalisAnthoceros agrestisKaffesäurederivate
DDC-Numbers
570
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Ernst, Lucien: Investigations on the formation of rosmarinic acid in Anthoceros agrestis and Melissa officinalis.. : Philipps-Universität Marburg 2024-12-09. DOI: https://doi.org/10.17192/z2024.0493.