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bioRxiv · 10.1101/2025.05.10.653210

Novel origin of replication for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Codik, A.·Kothari, A.·Liu, H.·Weinberg, B. L.·Owens, T. K.·Srinivasan, A.·Rivier, A.·Eng, T.·Arkin, A. P.·Deutschbauer, A. M.·Mukhopadhyay, A.
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Abstract

Plasmids isolated or characterized from environmental samples serve as a resource that can be used to develop genetic tools for characterizing recently isolated or less-studied microbes. In this report, we leveraged sequences from a previously characterized groundwater plasmidome and developed a screen to identify novel plasmid origins. Putative origin sequences were used to construct a barcoded plasmid library, which contained both known and newly predicted origins. This library was tested against a panel of representative bacterial strains and led to the identification of 3 novel origins that putatively replicate in gram-negative bacteria not previously associated with these origin sequences. We empirically validated one of the newly identified origins, 6911, to be functional in both the model bacterial strain, Escherichia coli BW25113, as well as in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium from the widespread Pantoea genus. We confirmed that a plasmid bearing origin 6911 as the sole origin could replicate and had a copy number of 9 ({+/-} 2) in Pantoea sp. MT58. We successfully used a plasmid based on the new origin to express the reporter protein GFP, and two non-native metabolite pathways for the natural product, indigoidine and the terpenoid compound, isoprenol. By pairing functional novel origins of replication to non-model organisms this pipeline can expand the tool kit for genetic manipulations of both model and less-studied bacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/653210v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@45689corg.highwire.dtl.DTLVardef@d6daeeorg.highwire.dtl.DTLVardef@1bf4d06org.highwire.dtl.DTLVardef@59ccfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure.C_FLOATNO We developed a host-origin pair identification pipeline by constructing a plasmid library containing both literature-sourced and computationally predicted origins of replication. By conjugating this library into non-model microbes, we identified functional origins in diverse bacteria, and could leverage these findings to develop genetically tractable hosts for microbial engineering. C_FIG

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Codik, A., Kothari, A., Liu, H., Weinberg, B. L., Owens, T. K., Srinivasan, A., Rivier, A., Eng, T., Arkin, A. P., Deutschbauer, A. M., Mukhopadhyay, A.. 2025-05-12. Novel origin of replication for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products. https://doi.org/10.1101/2025.05.10.653210

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