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

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.

CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

“…However, for 25 years it has remained the only non-native vector reported to be able to self-replicate in cyanobacteria (17). Recently, two small plasmids native to Synechocystis, pCA2.4 and pCB2.4, have been engineered for gene expression (33,73,77). The pANS plasmid (native to PCC 7942) has also been adapted as a replicative vector, but so far it has been only shown to function in PCC 7942 and Anabaena PCC 7120 (78).…”

Harnessing transcription for bioproduction in cyanobacteria

“…PCC 6803 (Armshaw et al ) and broad‐host‐range vectors based on the RSF1010 replicon (Xu et al , Guerrero et al ) have been exploited to express heterologous products. Increasing the copy number of the gene by plasmid integration has enhanced the yield (Angermayr et al , Armshaw et al , Ng et al ). The use of antibiotics for selection and maintenance of strains is commonplace, but pose further challenges and restrictions.…”

Evaluation of New Genetic Toolkits and Their Role for Ethanol Production in Cyanobacteria

“…Zang et al [41] also tested the impact of the length of the homologous sites and determined a positive correlation between the length of the homologous sites and the transformation efficiency. Different sites are used for genomic integration and show no phenotypic effect [31,39,42,43]. Besides genomic integration, different approaches for plasmid-based expression were attempted.…”

“…Besides genomic integration, different approaches for plasmid-based expression were attempted. On the one hand, the integration of expression cassettes into the native plasmids pCA2.4 and pCC5.2 were tested and resulted in an 8-to 20-fold higher reporter gene fluorescence compared to genomic integration [39,43]. On the other hand, self-replicating plasmids with different origins of replications as expression platforms were investigated.…”