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

Abstract

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.

Production of C2–C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies

“…Microcompartment is another smart system that has been used by microbes to address the problem of unstable or reactive intermediates by encapsulating pathway enzymes within a protein shell [ 76 ]. Pdu BMC is a natural microcompartment that functions in the 1,2-PDO degradation process [ 77 ]. Lee et al tried to construct an artificial BMC for 1,2-PDO synthesis by adding N-terminal targeting sequences derived from Pdu BMC to the enzymes of 1,2-PDO synthesis pathway in E. coli [ 78 ].…”

Apparent size and morphology of bacterial microcompartments varies with technique

“…Even modest engineering efforts can affect the size, shape, and morphology of MCPs. For example, knocking out or over-expressing different shell proteins leads to dramatic changes in the shape of MCPs, with many appearing to be long, hollow tubes [33][34][35][36][37]. As engineering efforts continue, it will become increasingly important to have a standardized set of tools for the field to determine and compare the size, shape, and morphology of engineered or altered MCPs across different studies.…”

De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments

“…However, aggregation may be problematic if the full potential of BMCs is to be realized and therefore alternative targeting mechanisms may facilitate the further development of BMC technology. Towards this goal researchers have recently used protein-interaction domains such as PDZ, GBD and SH3 to target fluorescent proteins to recombinant BMCs in the host C. glutamicum 35 .…”