title: 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.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany. Electronic address: m.baumgart@fz-juelich.de.
authors.forename: Meike
authors.initials: M
authors.lastname: Baumgart
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Huber
authors.affiliation: Institute of Complex Systems (ICS-4 (Cellular Biophysics)), Forschungszentrum Jülich, Jülich, Germany; Institute of Complex Systems (ICS-6 (Structural Biochemistry)), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Iman
authors.initials: I
authors.lastname: Abdollahzadeh
authors.affiliation: Institute of Complex Systems (ICS-4 (Cellular Biophysics)), Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gensch
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany. Electronic address: j.frunzke@fz-juelich.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Frunzke
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057486:Carbon Cycle; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000201:enzymology / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D044222:Halothiobacillus / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D005810:Multigene Family / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase
keywords: BMC; Bacterial microcompartments; Carbon fixation; Corynebacterium glutamicum; RuBisCO; Synthetic biology