title: Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.
abstract: During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Karin M
authors.initials: KM
authors.lastname: Münch
authors.affiliation: Centre for Mathematical Sciences, Technische Universität München, Garching/Munich, Germany Institute of Computational Biology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Müller
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Wienecke
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Bergmann
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Steffi
authors.initials: S
authors.lastname: Heyber
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Braunschweig, Germany r.muench@tu-bs.de.
authors.forename: Richard
authors.initials: R
authors.lastname: Münch
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany Braunschweig Integrated Centre of Systems Biology (BRICS), Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D001410:Bacillus megaterium / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins