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

Abstract

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.

Keywords: Gene dosage; Gene expression; Synechocystis PCC6803; Yellow fluorescent protein (YFP); pCA2.4.

Fig. 1

Determination of an optimal reporter gene in Synechocystis PCC6803; optimisation of integration into plasmid pCA2.4. a Growth rate comparison for Synechocystis PCC6803 strains expressing yellow fluorescent protein (YFP, UL006) and β-galactosidase (GAL, UL022) under the control of the constitutive Ptrc promoter in a chromosomal integration site (psbaII). Note: the strain constitutively expressing β-galactosidase displays significant growth retardation. b UL006 (YFP) 72 h culture on the left; UL022 (GAL) 72-h culture on the right. Note: the poor green colour of the strain constitutively expressing β-galactosidase. c Expression of both reporter genes under the constitutive Ptrc promoter results in a similar fold increase in expression measurements compared to wild-type [3.74 fold (YFP, au) versus 3.31 fold (GAL, Miller units)]. d PCR screening of Ptrc-YFP integration within the chromosomal psbaII site. PCR primers utilised flanked either side of the targeted site of integration (psbaII site) of the transformed construct Ptrc-YFP. Lower ~1 kb band represents WT and higher ~4 kb represents integration of cassette. Increasing kanamycin concentration and inclusion of glucose allowed rapid generation of a segregated YFP-expressing strain. e Integration of the same cassette within pCA2.4 required higher concentration of kanamycin and sub-culture to liquid media and inclusion of glucose prevented detectable integration. Note: + with glucose, - no glucose. Again note the PCR primers utilised flanked either side of the targeted site of integration (predicted pCA2.4 neutral site, Additional file 1: Figure S1). Lower ~1 kb band represents WT and higher ~4 kb represents integration of cassette

Fig. 2

Relative fluorescence levels of screened promoters and stability of modified pCA2.4 strains. a Optical density measurements at OD_730nm of growth of different YFP-expressing strains over 72 h. Chromosomally integrated (psbA2 locus) YFP strains UL006 (Ptrc-YFP), UL007 (Ptrc-YFP-E*) and UL008 (PpsbA2-YFP) grow similarly to WT. b Measurement of UL006, UL007 and UL008 YFP fluorescence levels after 72 h. The highest YFP fluorescence level detected was in UL006. Note 2 mM of the inducer theophylline was utilised to induce expression of the Ptrc riboswitch in UL007. c UL018 and UL006 strain stability after 80 days culturing. Lane 1 marker, lane 2 wild-type, lane 3 UL006 minus kanamycin, lane 4 UL006 plus kanamycin, lane 5 wild-type, lane 6 UL018 minus kanamycin, lane 7 UL018 plus kanamycin. The lower band indicates the presence of WT; higher band indicates construct integration. No evidence of the WT band in transconjugant strains was observed

Fig. 3

Stability of UL018 YFP fluorescence over 14 days without selective pressure. a Relative fluorescence measurements over 14 days for UL006 (chromosomal Ptrc-YFP) and UL018 (pCA2.4 Ptrc-YFP) with and without kanamycin addition. b Growth rate of UL006 (chromosomal Ptrc-YFP) and UL018 (pCA2.4 Ptrc-YFP) with and without kanamycin addition over the same 14-day period

Fig. 4

Confocal microscopy analysis of Synechocystis PCC 6803 WT, UL006 and UL018 cells. On the left hand side, representative images of cells enhanced for imaging of YFP, on the right hand side, the same representative images of cells enhanced for imaging of auto-fluorescence of Synechocystis PCC 6803

Fig. 5

Growth rate and YFP fluorescence levels from 3 sub-cultures of UL006 and UL018. a Relative fluorescence measurements of three sub-cultures of UL006 and UL018 taken after 6 days growth without selective pressure (no kanamycin). b Optical density measurements at OD_730nm of three sub-cultures of UL006 and UL018 taken at the same time point