This work successfully transformed UTEX 3222 by natural transformation, generating a large panel of fully segregated axenic strains and establishes the foundation for developing UTEX 3222 as a scalable platform for photosynthetic biomanufacturing.
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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This chapter presents a concise, reproducible protocol to increase transformation efficiency in F. nucleatum by systematically bypassing RM barriers, and can be adapted to additional F. nucleatum isolates once their methylation context is defined.
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