(449f) Improving Transformation Efficiencies in Synechococcus Sp. Via the Novel Combination of DNA Methylation and Plasmid Multimers

Cyanobacteria represent attractive biocatalysts due to their ability to photosynthetically fix CO₂ and convert it into biomass and value-added chemicals. Currently, however, our ability to engineer cyanobacterial metabolism continues to be limited by a lack of versatile and well-characterized genetic tools and associated methodologies. For instance, the delivery genes or pathways of interest is inherently limited by low transformation efficiencies, which slows the overall design-build-test-learn cycle and prohibits applications involving library screening, among others. The availability of self-replicating episomal plasmids can be particularly helpful in this regard, however such parts in cyanobacteria have largely been limited to the RSF1010 broad host range plasmid, which is difficult to work with due to its large size and low copy number, and shuttle vectors derived from native plasmids, whose delivery relies mainly upon conjugation. To help address the need for versatile and easy to use tools for engineering cyanobacteria, we have developed a multi-pronged approach to improve transformation of both self-replicating episomal and chromosomal integration vectors by the synergistic combination of both site-specific methylation and the generation of plasmid multimers. The first approach relies upon incorporation and methylation of the Highly Iterated Palindrome 1 (HIP1) sequence, which is found in virtually all cyanobacteria. HIP1 is an eight base pair sequence (consensus: GCGATCGC) that is highly abundant in cyanobacterial genomes, occurring on average every 1000 bp in Synechococcus sp. PCC 7002. Although the exact function(s) of HIP1 remains unknown, it is hypothesized to play a role in DNA recombination, repair, and/or maintenance of chromosome structure. In a previous study, it was shown that HIP1 methylation improved the efficiency of chromosomal integration via natural transformation in Synechocystis sp. PCC 6803, which was thought to be due to the bypass of a restriction system targeting the HIP1 sequence¹. If such were the case, in the traditional paradigm of restriction modification systems, removal or methylation of restriction sites would increase transformation into a host strain. However, on the contrary, here we show that the addition and methylation of HIP1 sites to virtually any plasmid is sufficient to increase its natural transformation efficiency in cyanobacteria. To demonstrate this, an E. coli strain was constructed to express an integrated copy of the HIP1 methyltransferase gene from Synechocystis sp. PCC 6803, and used as the host for amplifying and methylating plasmids for transformation into cyanobacteria. The number of included HIP1 sites on the cyanobacterial vector, their relative spacing, and absolute placement on integrative plasmids was first optimized. From this it was determined that incorporation of HIP1 sites directly flanking the outside of the homology arms resulted in the greatest increase in transformation efficiency. For integration vectors with long homology arms (500 bp), methylation of introduced HIP1 sites increased transformation efficiency ~3-fold, whereas for shorter homology arms (250 bp) a ~100-fold improvement was observed. Meanwhile, we have also demonstrated that replicating plasmids can be transformed into in cyanobacteria via natural transformation using plasmid multimers which can be generated via either in vitro or in vivo methods. Ultimately, two approaches can be synergistically combined to further increase transformation efficiencies in cyanobacteria. To make use of these methods, we have also developed and characterized a series of novel replicating plasmids for use with Synechcococcus sp. PCC 7002 and the newly discovered fast growing strain Synechococcus sp. PCC 11901. Finally, equipped with these novel tools/protocols we engineer both Synechococcus sp. PCC 7002 and PCC 11901 for photosynthetic production of sugars. Given the ubiquitous nature of the HIP1 site, the tools/methods developed here are expected to be readily extended to all other naturally transformable cyanobacteria.

References:

1.Wang B, Yu J, Zhang W, Meldrum DR. Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC 6803. Appl Environ Microbiol. 2015 Dec;81(24):8500-6. doi: 10.1128/AEM.02575-15. Epub 2015 Oct 9. PMID: 26452551; PMCID: PMC4644663.