Broad host range plasmids are a very useful tool employed by metabolic engineers to quickly insert non-endogenous proteins and measure their enzymatic activity in other host organisms. To harness their unique metabolic capabilities, less well-characterized organisms are being studied as alternative metabolic engineering chassis. These microbes unfortunately do not have known, stable non-endogenous plasmids, and as a result, investigations concerning the activity of heterologous enzymes in non-model organisms can be challenging and slow. To streamline engineering efforts more effectively with non-model organisms, the mechanics on plasmid stability must be understood.
Rhodopseudomonas palustris CGA009 (hereafter
R. palustris) is a non-model organism which contains a wide variety of compelling metabolic traits, including carbon and nitrogen fixation, lignin breakdown product and other aromatic catabolic pathways, and bioplastic and hydrogen . Over the years, our lab has performed extensive research detailing the bioproduct formation efficiency of R. palustris when catabolizing lignin substrates, as well as identifying the various synthetic biology tools that are effective with modifying this useful organism and optimizing vector parts (Alsiyabi et al., 2021; Brown et al., 2020, 2022; Immethun et al., 2021). This study investigates the mobilization protein MobV and its effect on plasmid retention in
R. palustris. Mobilization proteins participate in horizontal gene transfer by nicking plasmid DNA, allowing for rolling-circle-replication and conjugation, before rejoining plasmid DNA afterwards. Interestingly, in contrast to other relaxase enzymes which typically contain tyrosine active sites, this enzyme is homologous to a previously annotated mobilization protein from the streptococcal plasmid PMV158, which contains histidine amino acid active sites. In addition, the effect of these mutations on plasmid copy number and the transcription of the plasmid’s replication protein will be examined through qPCR and fluorescent protein expression via flow cytometry. The significance of this study is increasing the understanding of the mechanisms by which the mobilization protein ensures stable plasmid maintenance, which is critical for harnessing this bacterium’s extraordinary biochemical capabilities. This study will also advance methods by which heterologous proteins can be incorporated into non-model proteomes.
R. palustris. Mobilization proteins participate in horizontal gene transfer by nicking plasmid DNA, allowing for rolling-circle-replication and conjugation, before rejoining plasmid DNA afterwards. Interestingly, in contrast to other relaxase enzymes which typically contain tyrosine active sites, this enzyme is homologous to a previously annotated mobilization protein from the streptococcal plasmid PMV158, which contains histidine amino acid active sites. In addition, the effect of these mutations on plasmid copy number and the transcription of the plasmid’s replication protein will be examined through qPCR and fluorescent protein expression via flow cytometry. The significance of this study is increasing the understanding of the mechanisms by which the mobilization protein ensures stable plasmid maintenance, which is critical for harnessing this bacterium’s extraordinary biochemical capabilities. This study will also advance methods by which heterologous proteins can be incorporated into non-model proteomes.
Alsiyabi, A., Brown, B., Immethun, C., Long, D., Wilkins, M., & Saha, R. (2021). Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction. Metabolic Engineering, 68, 1–13. https://doi.org/10.1016/j.ymben.2021.08.008
Brown, B., Immethun, C., Wilkins, M., & Saha, R. (2020). Rhodopseudomonas palustris CGA009 polyhydroxybutyrate production from a lignin aromatic and quantification via flow cytometry. Bioresource Technology Reports, 11. https://doi.org/10.1016/j.biteb.2020.100474
Brown, B., Wilkins, M., & Saha, R. (2022). Rhodopseudomonas palustris: A biotechnology chassis. In Biotechnology Advances (Vol. 60). Elsevier Inc. https://doi.org/10.1016/j.biotechadv.2022.108001
Immethun, C., Kathol, M., Changa, T., & Saha, R. (2021). Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium <em>Rhodopseudomonas palustris</em> BioRxiv, 2021.11.01.466785. https://doi.org/10.1101/2021.11.01.466785
