Improving Synechocystis sp. PCC 6803 as a model organism

Matthew Allan Pope, Biotechnology And Biological Sciences Research Council (Great Britain), Patrik Jones, Peter Nixon
2021
Cyanobacteria are photoautotrophic prokaryotes capable of converting sunlight into chemical energy via oxygenic photosynthesis. Fast growth rates, ease of maintenance and swift mutant generation have resulted in certain cyanobacterial species becoming model organisms for photosynthesis researchers and biotechnologists. One species has garnered attention, being heralded as a "green E. coli": Synechocystis sp. PCC 6803 is a freshwater, unicellular and naturally transformable cyanobacterium with a
more » ... anobacterium with a fully sequenced and annotated genome. In recent years, Synechocystis mutants have demonstrated their efficacy as cellular-factories by yielding chemical commodities from sunlight and atmospheric carbon. However, certain traits of Synechocystis hinder those who wish to genetically and metabolically engineer this organism. Firstly, Synechocystis is polyploid, possessing multiple copies of its genome per cell. The time required to generate mutants is far greater than for its heterotrophic counterpart, Escherichia coli. Mutations introduced into the genome via heterologous DNA molecules must first present themselves in all genome copies before mutant phenotypes are revealed. Secondly, the list of characterised genetic parts known to function in Synechocystis is limited in comparison to those available for E. coli. Here I investigate the environmental and biochemical factors that influence polyploidy in Synechocystis. I demonstrate that a simple 10-fold reduction of phosphate in the conventional growth medium, BG-11, yields significantly fewer genome copy numbers per cell. From this I developed a novel natural transformation protocol that reduces the time required to obtain fully segregated mutants in Synechocystis. Furthermore, I show that DnaA, a conserved prokaryotic DNA replication initiator protein, is not essential for DNA replication in Synechocystis but does play an important physiological role in regulating polyploidy and DNA replication. Furthermore, I characterised the importance of DnaN, the β-subunit of the DNA pol [...]
doi:10.25560/89221 fatcat:tcyzroql55cixbpu4yk6u3khly