Impact of the carbon flux regulator protein pirC on ethanol production in engineered cyanobacteria

Abstract

Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms such as cyanobacteria, which are engineered to synthesize valuable products directly from CO₂ and sunlight. For example, strains of the model organism Synechocystis sp. PCC 6803 have been generated to produce ethanol. Here, we performed a study to prove the hypothesis that carbon flux in the direction of pyruvate is one bottleneck to achieve high ethanol titers in cyanobacteria. Ethanol-producing strains of the cyanobacterium Synechocystis sp. PCC 6803 were generated that bear mutation in the gene pirC aiming to increase carbon flux towards pyruvate. The strains were cultivated at different nitrogen or carbon conditions and the ethanol production was analysed. Generally, a clear correlation between growth rate and ethanol production was found. The mutation of pirC, however, had only a positive impact on ethanol titers under nitrogen depletion. The increase in ethanol was accompanied by elevated pyruvate and lowered glycogen levels indicating that the absence of pirC indeed increased carbon partitioning towards lower glycolysis. Metabolome analysis revealed that this change in carbon flow had also a marked impact on the overall primary metabolism in Synechocystis sp. PCC 6803. Deletion of pirC improved ethanol production under specific conditions supporting the notion that a better understanding of regulatory mechanisms involved in cyanobacterial carbon partitioning is needed to engineer more productive cyanobacterial strains.

Keywords: CO2; biofuel; carbon availability; green biotechnology; nitrogen limitation.

Figure 1

Experimental setup to evaluate the ethanol production in different strains of Synechocystis sp. PCC 6803. All strains were maintained on agar plates. Cells were freshly suspended into Erlenmeyer flasks and pre-cultured shaking at 50 μmol photons m⁻² s⁻¹ (μE) in complete BG11 under ambient air (0.04%CO₂, LC) for 1 week. Then, cells were grown in copper-free (-Cu) BG11 to induce expression of the ethanologenic cassette. Production assays were done in the Multi-Cultivator MD1000 in BG11-Cu at high CO₂ (5%, HC) and different light intensities of 250 or 500 μmol photons m⁻² s⁻¹ for 1 week. Sampling time points are indicated.

Figure 2

Ethanol production by different Synechocystis sp. PCC 6803 strains at 250 μmol photons m⁻² s⁻¹. Production assays were done in the Multicultivator MD1000 in BG11-Cu at high CO₂ (5%, HC). (A) Ethanol titer per culture volume. (B) Ethanol titer per biomass as OD₇₂₀. Mean values and standard deviations are shown (WT: n = 3; WT 219: n = 3; ∆pirC 219: n = 4).

Figure 3

Ethanol production by different Synechocystis sp. PCC 6803 strains at 500 μmol photons m⁻² s⁻¹. Production assays were done in the Multicultivator MD1000 in BG11-Cu at high CO₂ (5%, HC). (A) Ethanol titer per culture volume. (B) Ethanol titer per biomass as OD₇₂₀. Mean values and standard deviations are shown (n = 2).

Figure 4

Ethanol production by different Synechocystis sp. PCC 6803 strains at 500 μmol photons m⁻² s⁻¹ and reduced nitrogen content. Production assays were done in the Multicultivator MD1000 in BG11-Cu with 1.8 mM NaNO₃ (1/10 nitrate of the original BG11 medium) at high CO₂ (5%, HC). (A) Ethanol titer per culture volume. (B) Ethanol titer per biomass as OD₇₂₀. Mean values and standard deviations are shown (n = 4). Statistically significant differences between WT 219 and ∆pirC 219 are indicated with the respective p-values (significant when p ≤ 0.05).

Figure 5

Glycogen accumulation in different Synechocystis sp. PCC 6803 strains. The strains were cultivated in BG11-Cu at 250 μmol photons m⁻² s⁻¹ (WT: n = 3; WT 219: n = 3; ∆pirC 219: n = 4) (A), 500 μmol photons m⁻² s⁻¹ (WT: n = 2; WT 219: n = 2; ∆pirC 219: n = 2) (B), or at 500 μmol photons m⁻² s⁻¹ in BG11-Cu with 1.8 mM NaNO₃ (1/10 nitrate of the original BG11 medium, WT, n = 4, WT 219, n = 4, ∆pirC 219, n = 4) (C) for 7 days. Glycogen was quantified as glucose released by α-amylase from the cell pellets. Mean values and standard deviations are shown (n = 3). Statistically significant differences are indicated with the respective p-values (significant when p ≤ 0.05).

Figure 6

Metabolic changes of different Synechocystis sp. PCC 6803 strains during ethanol production. The strains were cultivated at 500 μmol photons m⁻² s⁻¹ in BG11-Cu with 1.8 mM NaNO₃ (1/10 nitrate of the original BG11 medium) for 7 days. With the exception of glycogen and ethanol, all metabolites were quantified by LC/MS-MS. Mean values and standard deviations are shown (n = 4). Statistically significant differences are indicated with the respective p-values (significant when p ≤ 0.05).