Book/Dissertation / PhD Thesis

FZJ-2020-02105

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Engineering of Corynebacterium glutamicum towards increased malonyl-CoA availability for polyketide synthesis

Milke, L. (Corresponding author)FZJ*

2020
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-480-5

Please use a persistent id in citations: http://hdl.handle.net/2128/25338  urn:nbn:de:0001-2020072314

Abstract: Polyketides are a structurally highly diverse group of natural products with interesting healthpromoting effects on humans. Despite all structural differences, polyketides are synthesized from simple CoA-activated carboxylic acid derivatives, such as acetyl-CoA or malonyl-CoA following a mechanism closely related to fatty acid biosynthesis. Unfortunately, polyketides are only synthesized in small quantities by the respective native organism. In contrast, microbial polyketide synthesis using engineered bacteria is a promising approach to get access to the desired products. Against this background, $\textit{Corynebacterium glutamicum}$ strains for the production of different plant polyketides such as stilbenes and flavonoids have been constructed recently. However, it soon became evident that the intracellular availability of malonyl-CoA is limiting the overall product formation in these strains. Hence, the main goal of this thesis was to optimize the intracellular malonyl-CoA availability in $\textit{C. glutamicum}$ by metabolic engineering. Additionally, the tailored strains should be used for establishing synthesis of biotechnological interesting polyketides. The following results were obtained: 1) Reduction of the citrate synthase activity to 5.5 % compared to the $\textit{C. glutamicum}$ wild type by exchanging the promotor of the encoding $\textit{gltA}$ gene, reduced acetyl-CoA consumption via the tricarboxylic acid cycle, which in turn improved malonyl-CoA availability. Upon transcriptional deregulation of $\textit{accBC}$ and $\textit{accD1}$ encoding the two subunits of acetyl-CoA carboxylase, malonyl-CoA synthesis from acetyl-CoA was drastically improved allowing for the synthesis of 65 mg/L (0.24 mM) naringenin und 450 mg/L (1.97 mM) resveratrol. Furthermore, improving the glucose uptake and elimination of anaplerotic pyruvate carboxylation reaction further contributed to an improved intracellular malonyl-CoA availability in the ultimately constructed strain $\textit{C. glutamicum}$ M-CoA. 2) Through episomal expression of genes encoding heterologous type III polyketide synthases from various plant species in the constructed strain $\textit{C. glutamicum}$ M-CoA, microbial synthesis of a pentaketide (noreugenin) but also phenylbutanoids (raspberry ketone, zingerone, benzylacetone) with a $\textit{ldhA}$-deficient variant could be established. The respective strains allowed for the synthesis of up to 53.3 mg/L (0.28 mM) noreugenin, 100 mg/L (0.61 mM) raspberry ketone, 70 mg/L (0.36 mM) zingerone and 10.5 mg/L (0.07 mM) benzylacetone from simple precursor molecules, respectively. 3) Hitherto, only type III polyketides can be synthesized by engineered $\textit{C. glutamicum}$ strains. In the context of this study, functional expression of a codon-optimized gene variant encoding the type I polyketide synthase 6-methylsalicylic acid synthase ChlB1 from $\textit {Streptomyces antibioticus}$ of 1,756 amino acids size was achieved. This allowed for the synthesis of up to 41 mg/L (0.27 mM) 6-methylsalicylic acid. It was found that $\textit{C. glutamicum}$ has an endogenous phosphopantetheinyltransferase activity, which can post-translationally activate ChlB1. This makes $\textit{C. glutamicum}$ a promising host for the production of other interesting type I polyketides.

Note: Biotechnologie 1
Note: Heinrich-Heine-Universität Düsseldorf, Diss., 2020

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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 2020

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Database coverage:
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