Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803

Abstract

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.

Figure 1. Work-flow for construction of the primary self-replicating wide-host-range vector pDF-trc.

The unique restriction sites (Table 1) for exchange of different genetic elements (Table 2) are shown in red.

Figure 2. The relationship between the amount of EFE and accumulation of ethylene in the headspace of closed cultures of E. coli.

E. coli DH5α harboring the pDF-trc-EFEh vector with P_trc was cultivated in LB medium and induced with various concentrations of added IPTG. (A) EFEh was purified by affinity chromatography and the amount of target protein was quantified using Image Lab software (BioRad) relative to the total protein content and the final OD₆₀₀ of the cultures. (B) The ethylene accumulation in closed E. coli DH5α cultivation vessels is plotted relative to the amount of recombinant EFE (in percentage relative to the amount of EFEh with maximum dose of IPTG) that was present in each vessel. Protein synthesis was induced by the addition of 25 µM or 1 mM IPTG that was added at an optical density of 0.1 (600 nm). Cultures to which no IPTG was added were used as controls. The concentration of IPTG that was added to each sample is shown in both panels.

Figure 3. The rate of ethylene synthesis in E. coli and Synechocystis in response to promoter choice and IPTG.

Two different plasmids were evaluated, pDF-trc-EFEh (P_trc), pDF-lac-EFEh (P_A1lacO-1). The + symbol indicates that expression was induced by the addition of 1 mM IPTG. Cultures to which no IPTG was added are indicated by n.a.

Figure 4. Topography of the lac derived promoters.

The -10 and -35 hexamers are highlighted in grey, and the lac operators are underlined. The predicted center of each operator is highlighted in red font. The transcriptional start site is highlighted with bold font and blue color.

Figure 5. The effect of IPTG concentration, promoter and cell density on the relative rate of ethylene synthesis in Synechocystis.

(A) The rate of ethylene synthesis with pDF-lac-EFEh (P_A1LacO-1) in response to the optical density of the culture and the presence or absence of IPTG. Open squares: No IPTG addition. Closed circles: 1 mM IPTG. (B) is identical to (A) with the exception that the rate of ethylene synthesis is shown relative to the optical density. (C) The rate of ethylene synthesis in response to IPTG concentration (logarithmic scale) with pDF-trc-EFEh (P_trc, black closed symbols), pDF-lac-EFEh (P_A1lacO-1, white open symbols). All cultures were measured at approximately the same optical density (OD₇₅₀ = 0.5). The inset graph differs only from the larger graph by having a linear scale on the X-axis.

Figure 6. The genomic structure of the native metal-inducible promoter elements.

The red line indicates the approximate fragment cloned for the construction of each of the pDF plasmids. The names of the generated vectors are indicated below each red line.

Figure 7. The rate of ethylene synthesis in E. coli and Synechocystis using three different metal-inducible promoters from cyanobacteria.

The rate of ethylene synthesis was analyzed in cultures of E. coli (light grey bars) and Synechocystis (dark grey bars) carrying the metal-inducible plasmids pDF-pet-EFEh (P_petE), pDF-coa-EFEh (P_coa) and pDF-smt-EFEh (P_smt). Cyanobaterial cultures were grown in BG-11 medium, lacking copper or cobalt for the evaluation of P_petE and P_coa, respectively. E. coli cells were grown in LB medium. The ethylene concentration was measured in closed vials that were either not induced (no addition) or induced using 0.5 µM of CuSO₄ for cells harbouring pDF-pet-EFEh, 6 µM of CoCl₂ for cells harbouring pDF-coa-EFEh, or 2 µM of ZnCl₂ for cells harbouring pDF-smt-EFEh.

Figure 8. The structure of the synthetic quorum-sensing induction systems used for the constructs (A) pDF-luxRI-EFEh and (B) pDF-rhlRI-EFEh.

Figure 9. The accumulation of ethylene (black line) and optical density (blue line) with either the (A) constitutive P_trc or (B) quorum-sensing P_luxRI promoters in E. coli.

Note that there is effectively no ethylene synthesis until OD₆₀₀ has reached approximately 0.5 with the quorum-sensing promoter. 10 mM FeCl₃ was added to the LB media in all cultures.