Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters

Abstract

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO₂ as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P _rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Keywords: Synechocystis; cyanobacteria; pSEVA plasmids; promoters; synthetic toolbox.

Figure 1.

Growth curves of Synechocystis wild-type (WT) and mutants harboring the replicative plasmids pSEVA251, pSEVA351 and pSEVA451. Cultures were grown at 30°C with rotary shaking (100 rpm) under (a) 12 h light (20 µE/m²/s)/12 h dark or (b) continuous light (20 µE/m²/s). Growth was monitored by measuring optical density at 730 nm (OD₇₃₀). Error bars correspond to standard deviations from three biological replicates with technical duplicates.

Figure 2.

Flow cytometry analysis of GFP fluorescence intensity in cells of Synechocystis wild-type (WT) and Synechocystis mutants harboring the empty plasmid pSEVA251 or the GFP generator BBa_E0240 under the control of two different promoters P_rnpB (reference promoter) and P_trc.x.lacO (later characterized in this study)—pSEVA251 P_rnpB::gfp (a and c) or pSEVA251 P_trc.x.lacO::gfp (b and d). The cells were grown in BG11 without or with kanamycin (Km). Histograms of GFP fluorescence intensities acquired in FL1 channel show the establishment of the GFP+ gates (black solid line) in Synechocystis mutants harboring the promoter P_rnpB (a) or P_trc.x.lacO (b) to evaluate the percentage of GFP+ cells (c and d). Histograms are representative of fluorescence intensity of cells analyzed after 16 days of cultivation; the error bars correspond to standard deviations from three biological replicates with technical duplicates. n.d. indicates non-detectable percentage of GFP+ cells for the WT and the strains harboring empty plasmid (d).

Figure 3.

Characterization of heterologous promoters in Synechocystis. (a) Schematic representation of the promoters and the GFP generator (BBa_E0240) assembly. (b) Normalized GFP fluorescence of Synechocystis cultures harboring the P_λcI, P_T7pol, P_araC, P_luxR and P_BADwt promoters. Measurements were performed up to three days (0, 24, 48 and 72 h) and the fluorescence was normalized to Abs_790. Fluorescence of the control strain harboring the pSEVA251 was subtracted from each sample. Error bars correspond to standard deviations from three biological replicates with technical triplicates (measured in duplicate). P_rnpB::gfp was included for comparison purposes.

Figure 4.

Characterization of the P_λcI regulated expression in Synechocystis. (a) Schematic representation of the promoter GFP generator (BBa_E0240) assembly followed by P_rnpB::ORF encoding the regulatory protein. (b) Normalized GFP fluorescence of Synechocystis cultures harboring the promoter P_λcI in presence (P_λcI::gfp-P_rnpB::cI) or in absence (P_λcI::gfp) of the CI repressor. Measurements were performed up to three days (0, 24, 48 and 72 h) and the fluorescence was normalized to Abs₇₉₀. The fluorescence of the control strain harboring the pSEVA251 was subtracted from each sample. Error bars correspond to standard deviations from three biological replicates with technical triplicates (measured in duplicate). P_rnpB::gfp was included for comparison purposes.

Figure 5.

Redesigned promoter sequences. (a) Promoter sequences of P_T7.1.x.lacO, P_T7.2.x.lacOand P_T7.3.x.lacO. The mutations are highlighted in red and the lacO operator in gray. The P_T7pol original promoter sequence was included for comparison purposes. (b) Promoter sequences of P_trc.x.lacO, P_trc.x.araO, P_trc.x.tetO1and P_trc.x.tetO2. The predicted -35 and -10 boxes are highlighted in purple, the lacO operator in gray, the lacOid operator in green, the araO operators in yellow, the tetO operator in light blue and the modified tetO operator in dark blue. The P_trc1O original sequence was included for comparison purposes.

Figure 6.

Characterization of the P_T7pol and redesigned P_T7pol promoter variants in Synechocystis constitutively expressing the T7 polymerase. Normalized GFP fluorescence of Synechocystis cultures harboring the P_T7pol, P_T7.1.x.lacO, P_T7.2.x.lacO and P_T7.3.x.lacO. Measurements were performed up to three days (0, 24, 48 and 72 h) and the fluorescence was normalized to Abs₇₉₀. The fluorescence of the control strain harboring the pSEVA251 was subtracted from each sample. Error bars correspond to standard deviations from three biological replicates with technical triplicates (measured in duplicate). P_rnpB::gfp was included for comparison purposes.

Figure 7.

Characterization of the hybrid P_tacI and the redesigned trc promoters in Synechocystis. (a) Normalized GFP fluorescence of Synechocystis cultures harboring the P_tacI, P_trc.x.tetO1, P_trc.x.araO, P_trc.x.tetO2 and P_trc.x.lacO. Measurements were performed up to three days (0, 24, 48 and 72 h) and the fluorescence was normalized to Abs₇₉₀. The fluorescence of the control strain harboring the pSEVA251 was subtracted from each sample. Error bars correspond to standard deviations from three biological replicates with technical triplicates (measured in duplicate). P_rnpB::gfp was included for comparison purposes. (b) Confocal micrographs of Synechocystis cells harboring the P_rnpB, P_trc.x.tetO2or P_trc.x.lacO promoters assembled with the GFP generator. Autofluorescence is depicted in the left column, GFP signal in the middle column and the merged signals in the right column. Scale bar, 2 µm.

Figure 8.

Characterization of the regulated expression of the P_trc.x.lacO in Synechocystis. Normalized GFP fluorescence was determined for cell cultures harboring the redesigned trc promoter and expressing the LacI repressor (P_trc.x.lacO::gfp-P_rnpB::lacI) without (-) or with addition (1 or 2 mM) of IPTG. Measurements were performed up to three days (0, 24, 48 and 72 h) and the fluorescence was normalized to Abs₇₉₀. The fluorescence of the control strain harboring the pSEVA251 was subtracted from each sample. Error bars correspond to standard deviations from three biological replicates with technical triplicates (measured in duplicate). Data from P_rnpB::gfp and P_trc.x.lacO::gfp were included for comparison purposes.