Construction of Recombinant Pdu Metabolosome Shells for Small Molecule Production in Corynebacterium glutamicum

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.