Heterologous expression of the Halothiobacillus neapolitanus carboxysomal gene cluster in Corynebacterium glutamicum

Abstract

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.

Bacterial microcompartments

“…Carboxysomes have also been the subject of extensive bioengineering efforts to enable or enhance CO 2 fixation. The cso operon encoding the α carboxysome of the chemoautotroph Halothiobacillus neapolitanus has been expressed in E. coli 121 and Corynebacterium glutamicum 122 , and the observed carboxysome-like particles were shown to have CO 2 fixing RuBisCO activity. The genes that encode for β-carboxysomal shell and core proteins have been expressed in the chloroplasts of Nicotiana benthamiana , resulting in the generation of carboxysome-like aggregates 123 .…”

Engineering and Modulating Functional Cyanobacterial CO2-Fixing Organelles

“…Furthermore, previous studies have demonstrated the possibilities of engineering entire PDU BMCs from Citrobacter freundii and EUT BMCs from Salmonella enterica in E. coli ( Parsons et al, 2008 , 2010 ; Choudhary et al, 2012 ). Expressing the α-carboxysome operon from a chemoautotroph Halothiobacillus neapolitanus has also led to the production of recombinant carboxysome-like structures with CO 2 fixation activity in E. coli ( Bonacci et al, 2012 ) and in a Gram-positive bacterium Corynebacterium glutamicum ( Baumgart et al, 2017 ). Apart from engineering BMCs in bacterial expression systems, efforts have been made to express β-carboxysome components in the chloroplasts of the model plant tobacco Nicotiana benthamiana .…”

Biotechnological Advances in Bacterial Microcompartment Technology

“…Similarly, the transfer of the a-carboxysome operon from H. neapolitanus to E. coli resulted in the appearance of icosahedral structures inside the host that were shown to have CO 2 fixation activity and resemble the wild type organelle [45]. More recently, the same a-carboxysome operon has been successfully transferred to the gram-positive organism C. glutamicum [46]. It has been proposed that the transfer of the CO 2 concentrating mechanism (CCM) from cyanobacteria to plants has the potential to not only increase global carbon fixation but also increase agricultural yields.…”