Bacterial microcompartments have significant potential in the area of industrial biotechnology for the production of small molecules, especially involving metabolic pathways with toxic or volatile intermediates. Corynebacterium glutamicum is an established industrial workhorse for the production of amino acids and has been investigated for the production of diamines, dicarboxylic acids, polymers and biobased fuels. Herein, we describe components for the establishment of bacterial microcompartments as production chambers in C. glutamicum. Within this study, we optimized genetic clusters for the expression of the shell components of the Citrobacter freundii propanediol utilization (Pdu) bacterial compartment, thereby facilitating heterologous compartment production in C. glutamicum. Upon induction, transmission electron microscopy images of thin sections from these strains revealed microcompartment-like structures within the cytosol. Furthermore, we demonstrate that it is possible to target eYFP to the empty microcompartments through C-terminal fusions with synthetic scaffold interaction partners (PDZ, SH3 and GBD) as well as with a non-native C-terminal targeting peptide from AdhDH (Klebsiella pneumonia). Thus, we show that it is possible to target proteins to compartments where N-terminal targeting is not possible. The overproduction of PduA alone leads to the construction of filamentous structures within the cytosol and eYFP molecules are localized to these structures when they are N-terminally fused to the P18 and D18 encapsulation peptides from PduP and PduD, respectively. In the future, these nanotube-like structures might be used as scaffolds for directed cellular organization and pathway enhancement.

Compartmentalization represents a ubiquitous principle used by living organisms to optimize metabolic flux and to avoid detrimental interactions within the cytoplasm. Proteinaceous bacterial microcompartments (BMCs) have therefore created strong interest for the encapsulation of heterologous pathways in microbial model organisms. However, attempts were so far mostly restricted to Escherichia coli. Here, we introduced the carboxysomal gene cluster of Halothiobacillus neapolitanus into the biotechnological platform species Corynebacterium gluta-micum. Transmission electron microscopy, fluorescence microscopy and single molecule localization microscopy suggested the formation of BMC-like structures in cells expressing the complete carboxysome operon or only the shell proteins. Purified carboxysomes consisted of the expected protein components as verified by mass spectrometry. Enzymatic assays revealed the functional production of RuBisCO in C. glutamicum both in the presence and absence of carboxysomal shell proteins. Furthermore, we could show that eYFP is targeted to the carboxysomes by fusion to the large RuBisCO subunit. Overall, this study represents the first transfer of an α-carboxysomal gene cluster into a Gram-positive model species supporting the modularity and orthogonality of these microcompartments, but also identified important challenges which need to be addressed on the way towards biotechnological application.

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