Improving Synechocystis sp. PCC 6803 as a model organism
File(s)
Author(s)
Pope, Matthew Allan
Type
Thesis or dissertation
Abstract
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 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 polymerase III holoenzyme. Finally, I developed a novel far-red light inducible gene expression system in Synechocystis by identifying a DNA element that regulates far-red light photosynthesis in the cyanobacterium Chroococcidiopsis thermalis PCC 7203. I constructed a novel strain of Synechocystis that regulates YFP gene-expression in response to far-red wavelengths of light.
Version
Open Access
Date Issued
2020-09
Date Awarded
2021-04
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Nixon, Peter
Jones, Patrik
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)