PLANT

The Restriction Barrier in Cyanobacteria Chassis

Cyanobacteria synthetic biology has broad applications ranging from pharmaceutical production to carbon capture and bioremediation [1]. Regardless of the great potential of cyanobacteria chassis, engineering them remains difficult in part due to the presence of restriction-modification (R-M) systems that target foreign DNA, lowering transformation efficiency [2]. Despite awareness of this barrier to transformation and the development of techniques to avoid R-M systems in cyanobacteria, R-M systems continue to make engineering cyanobacteria a difficult task [2]. To improve the situation, we are developing a software pipeline for avoiding the presence of restriction sites on foreign DNA to be introduced into a cyanobacteria chassis, thereby making cyanobacteria easier to engineer.

Overcoming the Restriction Barrier

Previous approaches at overcoming the barrier presented by R-M systems to engineering cyanobacteria have included methylating foreign DNA and the removal of restriction sites as identified via known restriction enzymes (e.g. those documented on the REBASE database) [2]

The former approach leverages the fact that cyanobacteria methylate their own DNA to protect it from their R-M systems; by being methylated, foreign DNA can escape recognition by restriction enzymes despite the presence of restriction sites [2]. Methylating foreign DNA presents difficulties, however, in that the specific methyltransferases that are cognate to the restriction enzymes used by the target cyanobacteria must be identified and used to methylate the foreign DNA [2].

The latter approach identifies parts of a foreign DNA sequence that would be restriction sites for the cyanobacteria’s R-M system, replacing them with synonymous sequences in coding regions [2]. Although this approach does allow for foreign DNA to escape recognition by known restriction enzymes, it has limitations in that it relies on information on known restriction enzymes in the target cyanobacteria being available; this information may be unavailable for non-model cyanobacteria species, as was the case for the strain of M.aeruginosa>that was the focus of our project.

Our solution to the restriction barrier combines the latter approach with the preexisting Stealth program that recognizes putative restriction sites in genomes based on underrepresented sequences [3]. The Chameleon project developed by our team takes putative restriction sites identified by Stealth and removes them from coding regions of plasmid sequences by replacing targeted codons with synonymous codons; thus, a plasmid that would be targeted by the cyanobacteria chassis's R-M system is able to avoid recognition, thereby improving transformation efficiency without altering function. This solution avoids reliance on documentation of the target cyanobacteria's restriction enzymes in that it only requires a sequenced genome from the target cyanobacteria, enabling its application to non-model species.

Application to our Cyanobacteria Chassis - Microcystis aeruginosa>

Our chassis, M.aeruginosa>, is a bloom-forming cyanobacteria that produces microcystin, a liver toxin with severe economic, environmental, and health effects in global water systems affected by M.aeruginosa blooms. Despite its natural competency [4], the difficulty of transforming M.aeruginosa is well-documented [5].

In our wet lab, we are using the Chameleon project along with a pair of plasmids, one without its restriction sites removed and the other processed by the Chameleon project to remove restriction sites, to demonstrate improved transformation efficiency with the use of the Chameleon project. By doing so, we will have not only provided empirical evidence supporting both the Stealth program and the Chameleon project, but also laid the foundation for our future work in creating a high transformation efficiency plasmid to knock out expression of microcystin-synthesizing genes in M.aeruginosa>.

Alongside our wet lab work, we have also begun to validate the Chameleon project in silico for our application to UTEX 2385 M.aeruginosa. First, by using Stealth to identify putative restriction motifs in our partial UTEX 2385 M.aeruginosa sequenced genome and checking these putative restriction sites against known restriction motifs in REBASE, we found that 70% of the known PCC 9806 M.aeruginosa> restriction motifs corresponded to one of our putative restriction motifs; this suggests that the majority of the putative restriction motifs we are working with are real restriction motifs. Second, by matching the putative restriction motifs to sequences on our unmodified pSPDY plasmid, we identified sites on the plasmid that were likely to be targeted by the R-M system of UTEX 2385 M. aeruginosa. After running the pSPDY plasmid through the Chameleon project to yield the modified pSPDY plasmid, we were able to remove the majority of putative restriction sites on the coding regions of the plasmid without altering any of the corresponding amino acid sequences. The in silico validation of the Chameleon project through comparisons to REBASE and modification of our plasmid is discussed in greater detail on the Engineering Page of our wiki.

Further establishing our software pipeline's utility in non-model species as compared to the REBASE approach, we are specifically working with the UTEX 2385 strain of M.aeruginosa, which has not been previously sequenced and whose strain-specific restriction sites are not documented. By sequencing the strain and demonstrating that transformation efficiency in UTEX 2385 M.aeruginosa> can be improved without prior documentation of its restriction enzymes, we will have substantiated a method of making cyanobacteria easier to engineer using only the target cyanobacteria's sequenced genome.

General Application to Cyanobacteria Chassis

M.aeruginosais far from the only species of cyanobacteria that has proven difficult to engineer due to the restriction barrier. Given the great diversity of cyanobacteria, which are estimated to include about 2000 species across 150 genera [6], it is important to develop generalized solutions to the restriction barrier to simplify foundational work that inhibits progressive engineering of cyanobacteria. The methylation approach requires the characterization of new methyltransferases corresponding to the target species' R-M system for each new species, while the REBASE approach necessitates the identification of specific restriction sites in each new species; thus, these approaches are not easily generalizable. By contrast, the Chameleon project requires only the target cyanobacteria's sequenced genome to remove restriction sites from plasmid sequences, enabling a highly generalizable approach.

It is our hope that by making the Chameleon project, we will have helped to broaden the useful tools available to future iGEM teams and other researchers engineering cyanobacteria chassis.

  [1]	A. Satta, L. Esquirol, and B. E. Ebert, “Current Metabolic Engineering Strategies for Photosynthetic Bioproduction 
        in Cyanobacteria,” Microorganisms, vol. 11, no. 2, p. 455, Feb. 2023, doi: 10.3390/microorganisms11020455.
  [2]	K. Stucken, R. Koch, and T. Dagan, “Cyanobacterial defense mechanisms against foreign DNA transfer and their impact 
        on genetic engineering,” Biol. Res., vol. 46, no. 4, pp. 373-382, 2013, doi: 10.4067/S0716-97602013000400009.
  [3]	S. Hu, S. Giacopazzi, R. Modlin, K. Karplus, D. L. Bernick, and K. M. Ottemann, “Altering under-represented DNA 
        sequences elevates bacterial transformation efficiency,” University of California, Santa Cruz, California, 
        Aug. 06, 2023.
  [4]	F. Nies, M. Mielke, J. Pochert, and T. Lamparter, “Natural transformation of the filamentous cyanobacterium 
        Phormidium lacuna,” PLOS ONE, vol. 15, no. 6, p. e0234440, Jun. 2020, doi: 10.1371/journal.pone.0234440.
  [5]	L. Zhao, Y. Song, L. Li, N. Gan, J. J. Brand, and L. Song, “The highly heterogeneous methylated genomes and 
        diverse restriction-modification systems of bloom-forming Microcystis,” Harmful Algae, vol. 75, pp. 87-93, 
        May 2018, doi: 10.1016/j.hal.2018.04.005.
  [6]	W. F. Vincent, “Cyanobacteria,” in Encyclopedia of Inland Waters, Elsevier, 2009, pp. 226-232. 
        doi: 10.1016/B978-012370626-3.00127-7.