Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry.

Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was the most tolerant to ethanol. Nevertheless, to implement a production process using recombinant strains, the bioethanol produced will be required to be continuously extracted from the culture media via a membrane-based technological process for example to prevent detrimental effects on the biomass. The results presented here are of significance in defining the maximum threshold for bulk ethanol concentration in production media.

Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production.

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 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. 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 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. 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 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.