SignificanceNonmodel bacteria have essential roles to play in the future development of biotechnology by providing new sources of biocatalysts, antibiotics, hosts for bioproduction, and engineered “living therapies.” The characterization of such hosts can be challenging, as many are not tractable to standard molecular biology techniques. This paper presents a rapid and automated methodology for characterizing new DNA parts from a nonmodel bacterium using cell-free transcription–translation. Data analysis was performed with Bayesian parameter inference to provide an understanding of gene-expression dynamics and resource sharing. We suggest that our integrated approach is expandable to a whole range of nonmodel bacteria for the characterization of new DNA parts within a native cell-free background for new biotechnology applications.

dDuring 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.

No abstract