Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced pr
PatriciaArmshaw1*, DawnCarey1, ConSheahan2 and J. Tony Pembroke1
1Molecular andStructural Biochemistry Laboratory, Department ofChemical andEnvironmental Sciences; Materials andSurface Science Institute, University ofLimerick, Limerick, Ireland
2Department of Design and Manufacturing Engineering, University of Limerick, Limerick, Ireland
Background: The use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of prod-uct is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for max-imising gene expression and subsequent product formation following genetic manipulation.
Results: A native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candi-date for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expres-sion vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.
Conclusions: A significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally inte-grated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integra-tion into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.Keywords:Yellow fluorescent protein (YFP), pCA2.4, Gene dosage, Gene expression, Synechocystis PCC6803.