Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803

Abstract

Background: Cyanobacteria can be metabolically engineered to convert CO₂ to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules.

Results: The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 ± 3.1 µg mL^-1 OD^-1 day^-1) compared to the control strain (6.4 ± 1.4 µg mL^-1 OD^-1 day^-1). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 ± 1.3 and 18.3 ± 3.3 µg mL^-1 OD^-1 day^-1, respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 ± 4.48 µg mL^-1 OD^-1 day^-1) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis.

Conclusions: This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.

Keywords: Acetyl-CoA; Cyanobacteria; Ethylene; Phosphoenolpyruvate carboxylase (PEPc); Phosphoenolpyruvate synthase (PPSA).

Fig. 1

Main carbon fixation metabolism in cyanobacteria. The blue and the green colours correspond to native and non-native enzymes in Synechocystis PCC 6803, respectively. ACc: acetyl-CoA carboxylase, Arg: arginine, Aza: azaserine, Calvin cycle: Calvin–Benson–Bassham cycle, Chl a: chlorophyll a, CS: citrate synthase, Cyclo: cycloxydim, DHA-P: dihydroxyacetone phosphate, Efe: ethylene-forming enzyme, GS-GOGAT: glutamine synthase glutamine oxoglutarate aminotransferase, MDH: malate dehydrogenase, ME: malic enzyme, PEPc: phosphoenolpyruvate carboxylase, PPSA: phosphoenolpyruvate synthase, TCA cycle: tricarboxylic acid cycle

Fig. 2

Ethylene production by the Synechocystis strains created in this study (Table 2) cultivated under the standard treatment (BG11+50 mM Tris pH 8.0, 50 mM NaHCO₃, Km (25 µg mL⁻¹) Cm (20 µg mL⁻¹), 5 µM of NiCl₂ and 20 µE m⁻² s⁻¹). Asterisks correspond to statistical significant differences compared to CD-C and CD-P, respectively. The experiment was repeated three times with three biological replicates. Error bars represent the mean ± SE

Fig. 3

Ethylene production by the Synechocystis strain CD-P in different treatments. The different treatments were BG11, BG11₀, BG11+arginine, BG11₀+arginine, BG11+azaserine, BG11+cycloxydim, BG11₀+cycloxydim and BG11+Arg+azaserine+cycloxydim (Table 3) all containing 50 mM Tris pH 8.0, 50 mM NaHCO₃, Km (25 µg mL⁻¹), Cm (20 µg mL⁻¹), 5 µM of NiCl₂ and 20 µE m⁻² s⁻¹. Asterisks correspond to statistically significant differences. The experiment was repeated two times with three biological replicates. Mean ± SE

Fig. 4

Ethylene production of the Synechocystis strains, CD-C, CD-P, CD-P1 and CD-P4 in standard and BG11+cycloxydim treatment (Table 3). Asterisks correspond to statistical significant differences. The experiment was repeated twice with three biological replicates. Mean ± SE

Fig. 5

SDS-PAGE/Western immunoblot of the Synechocystis strains created in this study (Table 2) analysed for the presence of phosphoenolpyruvate synthase (PPSA) and ethylene-forming enzyme (EFE). A SDS-PAGE loaded with 3 µg of protein crude extract from the Synechocystis strains created in this study under standard treatment (Table 2) and B Western immunoblot using anti-Flag antibody for the Synechocystis engineered strains (Table 2) under standard treatment. Standard treatment is BG11+ 50 mM Tris pH 8.0, 50 mM NaHCO₃, Km (25 µg mL⁻¹), Cm (20 µg mL⁻¹) and 5 µM of NiCl₂ and 20 µE m⁻² s⁻¹. The upper band corresponds to PPSA (approximate molecular weight 91.17 and 92.56 kDa for PPSA₆₈₀₃ and PPSA₇₀₀₂, respectively) and the lower band to EFE (approximate molecular weight 42.16 kDa)

Fig. 6

SDS-PAGE/Western immunoblot of the Synechocystis strains created in this study (Table 2) analysed for the presence of ethylene-forming enzyme (EFE). A SDS-PAGE loaded with 3 µg of protein crude extract from the Synechocystis engineered strain CD-P in different treatments (Table 3) and B Western immunoblot using anti-Flag antibody from the Synechocystis engineered strain CD-P in different treatments tested (Table 3). The different treatments were BG11, BG11₀, BG11+arginine, BG11₀+arginine, BG11+azaserine, BG11+cycloxydim, BG11₀+cycloxydim and BG11+arginine+azaserine+cycloxydim (Table 3) all containing 50 mM Tris pH 8.0, 50 mM NaHCO₃, Km (25 µg mL⁻¹), Cm (20 µg mL⁻¹), 5 µM of NiCl₂ and 20 µE m⁻² s⁻¹

Fig. 7

SDS-PAGE/Western immunoblot of the Synechocystis strains created in this study (Table 2) analysed for the presence of phosphoenolpyruvate carboxylase (PEPc). A SDS-PAGE loaded with 31 µg of protein crude extract from Synechocystis strains created in this study under standard treatment (Table 2) and B Western immunoblot using anti-PEPc for the Synechocystis engineered strains (Table 2) in standard treatment. Standard treatment is BG11+ 50 mM Tris pH 8.0, 50 mM NaHCO₃, Km (25 µg mL⁻¹), Cm (20 µg mL⁻¹), 5 µM of NiCl₂ and 20 µE m⁻² s⁻¹. PEPc₆₈₀₃ and PEPc₇₀₀₂ corresponds to purified protein (100 ng loaded) of PEPc from Synechocystis PCC 6803 and Synechococcus PCC 7002, respectively