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bioRxiv · 10.64898/2026.09.15.751560

A methylome-guided toolkit for genetic engineering of the fast-growing cyanobacterium UTEX 3222

Lin, D.·Booth, A.·Gill, A.·Aung, M. M.·Kim, D.·Sagaram, U. S.
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Abstract

Cyanobacterium aponinum UTEX 3222 is a fast-growing marine cyanobacterium that reaches high cell densities, making it an attractive chassis strain for low-cost biomanufacturing. However, past efforts to genetically engineer UTEX 3222 have been unsuccessful. We hypothesized that the complex restriction modification systems in the UTEX 3222 genome constitute a major barrier to genetic transformation. Using a combined methylation avoidance and methylation mimicking approach, we overcame this barrier and successfully transformed UTEX 3222 by natural transformation, generating a large panel of fully segregated axenic strains. Transformation method was also established for a novel shuttle vector that replicates in UTEX 3222. A large-scale promoter screen identified promoters which exhibited strong expression at high cell density. These advances establish the foundation for developing UTEX 3222 as a scalable platform for photosynthetic biomanufacturing.

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Lin, D., Booth, A., Gill, A., Aung, M. M., Kim, D., Sagaram, U. S.. 2026-09-17. A methylome-guided toolkit for genetic engineering of the fast-growing cyanobacterium UTEX 3222. https://doi.org/10.64898/2026.09.15.751560

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