Book/Dissertation / PhD Thesis

FZJ-2017-05533

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Establishment of Bacterial Microcompartments in the Industrial Production Strain Corynebacterium glutamicum

Huber, I. (Corresponding author)FZJ*

2018
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-95806-302-0

Please use a persistent id in citations: http://hdl.handle.net/2128/19036  urn:nbn:de:0001-2018062802

Abstract: Bacterial microcompartments (BMCs) have significant potential in the area of industrial biotechnology for the production of small molecules with toxic or volatile intermediates. $\textit{Corynebacterium glutamicum}$ is an established industrial platform organism for the production of amino acids and was made accessible for the production of diamines, dicarboxylic acids, polymers and bio-based fuels. The aim of this study was to establish BMC production in $\textit{C. glutamicum}$ and to provide useful tools towards a biotechnological application of BMCs as nano-bioreactors. Within this study, optimized gene clusters for the expression of the $\textit{Citrobacter freundii}$ propanediol utilization compartment shell genes were constructed. Upon induction in $\textit{C. glutamicum}$, transmission electron microscopy images revealed heterologous compartment production and assembly in the mid cell. Growth studies demonstrated a drastic negative impact of BMC production in $\textit{C. glutamicum}$ but reasonable results were obtained with the chromosomal integration of the gene cluster. To evaluate the potential of BMCs in $\textit{C. glutamicum}$, the methanol consumption and the ethanol production pathway, which both include a toxic aldehyde intermediate, and the itaconate production pathway including a transient intermediate, were used. One issue, however, was to produce the enzymes tagged with N-terminal encapsulation peptides in an active form. For the methanol consumption pathway, the enzyme Hps was inactive and also AdhB for ethanol production and MalECad for itaconate production showed reduced activities of 17% and 35% respectively. To expand the synthetic repertoire of encapsulation peptides, new strategies to target proteins of interest into the compartment lumen were investigated. Using fluorescence microscopy, it was proven that a none-native C-terminal targeting peptide from $\textit{Klebsiella pneumonia}$ and three synthetic-scaffolding peptides are able to localize a fluorescence reporter into the compartment lumen. The establishment of the alternative the C-terminal targeting strategy now offers the opportunity to choose the optimal fusion for the particular protein of interest. This was, for example demonstrated with C-terminally targeted AdhB versions, restoring an 18% higher enzymatic activity than the N-terminally targeted AdhB version. PduA formed bundles of filaments in $\textit{C. glutamicum}$ and encapsulation peptide tagged fluorescence reporters localized to those structures. This demonstrates that there is the opportunity to use the nanotube-like structures as scaffolds for directed cellular organization and pathway enhancement. Altogether, this work provides essential fundamental groundwork on heterologous BMC or scaffold formation in $\textit{C. glutamicum}$ and paves the way for metabolic engineering of pathways with toxic or volatile intermediates or pathways with competing reactions.

Note: Biotechnologie 1
Note: Universität Düsseldorf, Diss., 2017

---

Contributing Institute(s):

1. Biotechnologie (IBG-1)

Research Program(s):

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

Appears in the scientific report 2017

---

Database coverage:
;

---