Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942

Abstract

Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from photosynthetic CO2 fixation or from remobilisation of existing carbon stores.

Keywords: biotechnology of microorganisms; cyanobacteria; nitrogen deprivation; overflow metabolism; photosynthesis; physiolology and metabolism; pyruvate excretion.

FIGURE 1

Physiological assessment of Synechococcus elongatus WT and ΔglgC cultures under N-starvation. The (A) culture density (A₇₃₀), (B) carbohydrate : amide II ratio, (C) net oxygen evolution, (D) ratio of phycocyanin to optical density, (E) chlorophyll a concentration, and (F) extracellular pyruvate concentration of S. elongatus WT and ΔglgC cultures over 96 h nitrogen starvation. Data indicate mean ± SE (n = 4) for four biological replicates.

FIGURE 2

Extracellular pyruvate concentration in cultures of S. elongatus ΔglgC after 48 h N-deprivation against net oxygen evolution. Pyruvate levels are shown (A) pre-N deprivation, (B) 2 h after N-deprivation, and (C) 24 h after N-deprivation. Results represent a combination of multiple experiments. Pyruvate concentration is normalized to 1 OD₇₃₀ at 0 h with an OD₇₃₀ range of 0.84–1.2. Each point is displayed as the average of technical duplicates from pooled sets of experiments.

FIGURE 3

Extracellular concentration of pyruvate, and net oxygen evolution at different light intensities for S. elongatus ΔglgC after 48 h in N-deprivation culture. Data points represent mean ± SE (n = 3; the average of two technical duplicates for three biological replicates).

FIGURE 4

Extracellular concentration of pyruvate in S. elongatus 7942 ΔglgC, ΔglgC+galP, and ΔglgC+cscKB after 48 h N-deprivation and photomixotrophic growth. Data indicate mean ± SE (n = 3; the average of two technical duplicates for three biological replicates).

FIGURE 5

The relationship between extracellular pyruvate at 48 h N-deprivation against the density of the starter culture density of S. elongatus ΔglgC just prior to dilution to a standard 1 OD₇₃₀ at time zero. Data points displayed as the average of technical duplicates from pooled sets of experiments.

FIGURE 6

The effect of pH on extracellular pyruvate levels. Growth ΔglgC under N-deprivation at high pH increases extracellular pyruvate. The extracellular concentration of pyruvate was measured over 96 h of N-deprivation at different media pH. Cultures were standardized to 1 OD₇₃₀ at 0 h N-deprivation for the pH8 and pH10 treatments. Displayed are the averages of technical duplicates of biological quadruplicates. Data points indicate mean ± SE (n = 4).