Abstract
BackgroundThe 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 product 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 maximising gene expression and subsequent product formation following genetic manipulation.ResultsA 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 candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression 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.ConclusionsA 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 integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0385-x) contains supplementary material, which is available to authorized users.
Highlights
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
While both yellow fluorescent protein [YFP] and β-galactosidase (GAL) expression were detectable at similar levels with a chromosomal integration (Fig. 1c), it was found that integration of the β-galactosidase gene within the chromosomal neutral site resulted in significant growth retardation (Fig. 1a)
Integration of a β-galactosidase expressing construct into pCA2.4 could lead to even higher expression levels, it was determined that YFP, which displayed a similar growth rate to wild-type when expressed under the control of the Ptrc promoter within the chromosomal neutral site would be a more favourable reporter gene to be utilised throughout this analysis
Summary
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. Reserves of fossil fuels are declining while greenhouse gas emissions are on the rise [1] This has led to an increasing emphasis on the research of biofuel production systems that harness CO2. Strain PCC6803, referred to as Synechocystis PCC6803, is commonly used as a model cyanobacterium due to its relative ease of genetic manipulation; its ability to grow both autotrophically and heterotrophically with minimal light; and ability to undergo natural transformation [5]. This strain can express several well-characterised Escherichia coli promoters, with altered control in many cases [6]. The issue of initial low production levels of target compounds of interest is a limiting concern [9]
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