Exploration and evaluation of novel restriction-modification systems in the edible, filamentous cyanobacterium Arthrospira platensis NIES-39
Year: 2025
Type: preprint
Abstract: Arthrospira platensis (spirulina) is an edible cyanobacterium with significant potential as a sustainable platform for biomanufacturing and in situ resource utilization in space exploration. However, its genetic tractability has been limited by the large and poorly characterized repertoire of restriction-modification (R/M) systems that degrade foreign DNA. To overcome this barrier, we explored and functionally evaluate novel R/M systems in A. platensis NIES-39. Using Pacific Biosciences single-molecule real-time sequencing and bioinformatics analysis, we identified six distinct methylated DNA motifs and several candidate Type II R/M systems. Putative methyltransferase (MTase) genes were selected for validation through heterologous expression in Escherichia coli . In vitro assays with purified recombinant protein and in vivo plasmid protection assays successfully demonstrated the activity of four MTases, M. HgiCI, M.PstI, M.Tth111I, and M. HgiDI, which protect against cognate restriction enzymes. Furthermore, co-expression of these MTases in E. coli protected target plasmids from cleavage, providing a practical method for preparing DNA for transformation. Characterizing these active R/M systems and validating their methyltransferases provide crucial insights and molecular tools to improve genetic engineering in A. platensis . These findings will accelerate development of this cyanobacterium as a robust chassis for synthetic biology applications, ranging from enhanced nutrition to on-demand pharmaceutical production.
Authors: Shunsuke Yamazaki, Jacob M. Hilzinger, Skyler Freidline, Jeffrey Skerker, Adam Arkin
Institution: University of California, Berkeley
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W7089612742
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