Book/Dissertation / PhD Thesis

FZJ-2019-03123

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Combinatorial Biosynthesis of Natural and Non-natural Plant-derived Phenols in Microorganisms

Aschenbrenner, J. (Corresponding author)FZJ*

2019
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-419-5

Please use a persistent id in citations: http://hdl.handle.net/2128/22856  urn:nbn:de:0001-2019100910

Abstract: Phenylpropanoids and phenylpropanoid-derived plant polyphenols have numerous applications in pharmaceutical and food industries and are used for example as antibiotics, therapeutics and colourants. Unfortunately, their extraction from plants is not efficient due to low product concentrations. In addition, downstream processing is impeded because the desired phenylpropanoids are present in a complex mixture of compounds with very similar chemical properties. These limitations can be overcome using microbial platform organisms to produce phenylpropanoids and polyphenols. The following results were obtained in this thesis: (1) The efficient production of monolignols is an important requirement for the synthesis of the pharmacologically relevant lignans, which belong to the class of plant polyphenols. An engineered $\textit{Escherichia coli}$ strain, equipped with a set of genes encoding enzymes for monolignol synthesis, was used to elucidate the microbial production of different monolignols. For this purpose, natural and non-natural phenylpropanoids were supplemented to respective biotransformations using the engineered strain to examine the promiscuity of heterologous enzymes. It was revealed that the engineered strain was able to catalyse the stepwise reduction of six naturally occurring phenylpropanoids including 5-hydroxyferulic acid and sinapic acid, which was reported for the first time. Additionally, chemically interesting non-natural phenylpropanoids, such as 3,4,5-trimethoxycinnamic acid, 5-bromoferulic acid, 3-nitroferulic acid, and a bicyclic $\textit{p}$-coumaric acid derivative were also reduced to the corresponding non-natural monolignols. The microbial production of these compounds is a good basis for the synthesis of more complex plant derived compounds. (2) The site-specific and stereospecific decoration of phenylpropanoids with hydroxyl and O-methyl groups is a good possibility to obtain a variety of different phenylpropanoids. First a 4-coumarate 3-hydroxylase (C3H) from $\textit{Saccharothrix espanaensis}$ was used for the hydroxylation of $\textit{p}$-coumaric acid yielding caffeic acid. In a second step, this enzyme was combined with the previously used monolignol pathway to produce caffeoyl alcohol from supplemented $\textit{p}$-coumaric acid. Biotransformations resulted not only in caffeoyl alcohol production but also in $\textit{p}$-coumaryl alcohol production. This underlined the challenges of selective product synthesis with promiscuous enzymes. Further substitutions at the phenyl ring were introduced with the caffeic acid O-methyltransferase (COMT) from $\textit{Medicago sativa}$, which was engineered towards the methylation of caffeic acid and 5-hydroxyferulic acid in whole cell biotransformations with engineered $\textit{E. coli}$ strains. COMT libraries obtained from site-saturation mutagenesis at four positions were screened and selected strains were further characterised. Amino acid substitutions in four positions located in or adjacent to the methoxy binding pocket were shown to alter the substrate specificity towards caffeic acid and 5-hydroxyferulic acid to produce ferulic acid and sinapic acid, respectively. Variants were found with an increased productivity of up to 17 % for ferulic acid production and 46 % for sinapic acid production compared to the not engineered wild type $\textit{E. coli}$ strain. The results contribute to a better understanding of these enzymes and will help to perform metabolic engineering for the microbial production of more complex phenylpropanoid-derived compounds in the future. (3) The multicopper oxidase CueO from $\textit{Rhodococcus erythropolis}$, previously used for the $\textit{in vitro}$ production of lignan, was now examined regarding lignan production using a respective recombinant $\textit{E. coli}$ strain. The gene $\textit{cueO}$ was expressed under the control of a T7 expression system and a pBAD expression system to find the best suited induction conditions. It was revealed that the tight gene regulation of the pBAD expression system can be used for the microbial lignan production. The strain was cultivated in the presence of coniferyl alcohol (1 mM) and produced 0.17 mM (+)-pinoresinol. This is the first time, that microbial (+)-pinoresinol production was observed.

Note: Biotechnologie 1
Note: Dissertation, Heinrich-Heine-Universität Düsseldorf, 2019

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Contributing Institute(s):

1. Biotechnologie (IBG-1)

Research Program(s):

1. 581 - Biotechnology (POF3-581) (POF3-581)

Appears in the scientific report 2019

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Database coverage:
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