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Minicells as a potential chassis for engineering lineage-agnostic organisms

By Eric Wei, Anton Jackson-Smith, Drew Endy

Posted 02 Aug 2020
bioRxiv DOI: 10.1101/2020.07.31.231670

Background: Genomes encode for organisms and thus genome synthesis implies the possibility of organismal synthesis, including the synthesis of organisms without constraint to lineage. Current genome-scale engineering projects are focused on minimization, refactoring, or recoding within the context of existing natural lineages. Minicells arise naturally as anucleate cells that are devoid of heritable genetic material but are capable of gene expression. Thus, minicells may serve as a useful starting point for developing lineage-agnostic organisms encoded by newly-designed synthetic genomes. However, the composition and expression capacity of minicells is fixed at the time of their formation. The possibility of reestablishing cellular growth and division starting from minicells and entirely heterologous synthetic genomes is unknown. Results: We observed expression and segregation of functional proteins among mixed populations of reproducing cells and so-derived anucleate minicells via fluorescence microscopy. By adapting and integrating established methods of preparation and purification we were able to isolate minicells from a growing population of progenitor cells with a purity of at least 500 minicells per progenitor. We then used heterologous expression of plasmid-encoded green fluorescent protein to estimate the absolute expression capacity of minicells. We found that minicells can support the formation of 4.9 +/- 4.6 1E8 peptide bonds prior to exhausting their initial intrinsic expression capacity. Conclusions: Minicells can be produced in large numbers with high purity and can also harbor and express engineered plasmids. The observed variation in minicell gene expression capacity suggests that about 13% of gene-expressing minicells can support the formation of more than one-billion peptide bonds, an amount sufficient to replicate known prokaryotic proteomes. Stated differently, while most minicells would require a sophisticated genetic boot program to first increase minicell-specific expression capacity sufficient to instantiate newly reproducing lineages, a subset of minicells may be able to directly support whole genome, lineage-agnostic organism engineering. ### Competing Interest Statement The authors have declared no competing interest.

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