This title appears in the Scientific Report :
2018
Please use the identifier:
http://hdl.handle.net/2128/17923 in citations.
Corynebacterium glutamicum – a novel platform for the production of plant polyphenols
Corynebacterium glutamicum – a novel platform for the production of plant polyphenols
Plants synthesize a large diversity of more than 200,000 different secondary metabolites, which can be subdivided into terpenoids, alkaloids and polyphenols. Especially polyphenols demonstrate important pharmacological activities. Unfortunately, these compounds are produced only in small amounts ren...
Saved in:
Personal Name(s): | Kallscheuer, Nicolai (Corresponding author) |
---|---|
Contributing Institute: |
Biotechnologie; IBG-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2018
|
Physical Description: |
X, 98 S. |
Dissertation Note: |
Universität Düsseldorf Diss., 2017 |
ISBN: |
978-3-95806-291-7 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Addenda |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien / Key Technologies
160 |
Link: |
OpenAccess OpenAccess |
Publikationsportal JuSER |
Plants synthesize a large diversity of more than 200,000 different secondary metabolites, which can be subdivided into terpenoids, alkaloids and polyphenols. Especially polyphenols demonstrate important pharmacological activities. Unfortunately, these compounds are produced only in small amounts rendering the extraction from plant material economically not viable. The natural synthesis of polyphenols such as stilbenes and flavonoids starts from phenylpropanoids, which in turn are obtained from aromatic amino acids. The main goal of this thesis was to exploit the yet untapped potential of $\textit{Corynebacterium glutamicum}$, an industrial workhorse for amino acid production, for the synthesis of plant polyphenols by functional integration of heterologous pathways. To this end, the following results were obtained: (1) Initial attempts to produce the stilbene resveratrol from the phenylpropanoid $\textit{p}$-coumaric acid in $\textit{C. glutamicum}$ failed. It turned out that an unknown degradation pathway allowed $\textit{C. glutamicum}$ to grow with phenylpropanoids as sole carbon and energy source. The expression of a gene cluster coding for enzymes of unknown function was highly upregulated in presence of phenylpropanoids. It could be shown that the enzymes catalyze a CoAdependent, β-oxidative side chain shortening of phenylpropanoids yielding benzoic acids, which are further degraded by well-characterized pathways in $\textit{C. glutamicum}$. In addition to the identified pathway, the complex network for the degradation of aromatic compounds in $\textit{C. glutamicum}$ interfered with the desired production of polyphenols. To this end, a novel platform strain unable to degrade aromatic compounds (designated $\textit{C. glutamicum}$ DelAro$^{4}$) was constructed by deleting altogether 21 genes in four clusters in the genome. (2) Plasmid-borne expression of genes coding for heterologous enzymes from different plant species enabled production of stilbenes (pinosylvin, resveratrol and piceatannol) as well as of (2S)-flavanones (naringenin and eriodictyol) depending on the supplemented phenylpropanoid in different strains derived from $\textit{C. glutamicum}$ DelAro$^{4}$. Stilbene and (2S)-flavanone titers of up to 158 mg/L and 37 mg/L could be achieved, respectively. The production of O-methylated stilbenes and of more complex flavonoids could be demonstrated by introduction of additional genes. The obtained product titers of the flavonols quercetin (10 mg/L) and kaempferol (23 mg/L) exceeded the highest titers produced in engineered microorganisms so far. (3) Microbial production of resveratrol and naringenin with $\textit{C. glutamicum}$ could be achieved starting from glucose by deregulation of the shikimate pathway to achieve intracellular accumulation of aromatic precursor amino acids. A heterologous tyrosine ammonia lyase (TAL) connected the endogenous aromatic amino acid metabolism to the production of phenylpropanoid-derived polyphenols. The TAL activity was rate-limiting during polyphenol production from glucose. To circumvent the bottleneck at the stage of phenylpropanoid synthesis, a novel synthetic pathway for the production of the direct polyphenol precursor $\textit{p}$-coumaroyl-CoA starting from the cheap 4-hydroxybenzoic acid was first designed $\textit{in silico}$ and then functionally introduced in $\textit{C. glutamicum}$. The pathway is the non-natural reversal of a bacterial degradation pathway for phenylpropanoids and requires only acetyl-CoA and cofactors such as ATP and NADH. Initially, 5 mg/L resveratrol could be produced from 4-hydroxybenzoic acid using this novel pathway. |