Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism

Abstract

Succinate produced by microorganisms can replace currently used petroleum-based succinate but typically requires mono- or poly-saccharides as a feedstock. The cyanobacterium Synechocystis sp. PCC6803 can produce organic acids such as succinate from CO₂ not supplemented with sugars under dark anoxic conditions using an unknown metabolic pathway. The TCA cycle in cyanobacteria branches into oxidative and reductive routes. Time-course analyses of the metabolome, transcriptome and metabolic turnover described here revealed dynamic changes in the metabolism of Synechocystis sp. PCC6803 cultivated under dark anoxic conditions, allowing identification of the carbon flow and rate-limiting steps in glycogen catabolism. Glycogen biosynthesized from CO₂ assimilated during periods of light exposure is catabolized to succinate via glycolysis, the anaplerotic pathway, and the reductive TCA cycle under dark anoxic conditions. Expression of the phosphoenolpyruvate (PEP) carboxylase gene (ppc) was identified as a rate-limiting step in succinate biosynthesis and this rate limitation was alleviated by ppc overexpression, resulting in improved succinate excretion. The sugar-free succinate production was further enhanced by the addition of bicarbonate. In vivo labeling with NaH¹³CO₃ clearly showed carbon incorporation into succinate via the anaplerotic pathway. Bicarbonate is in equilibrium with CO₂. Succinate production by Synechocystis sp. PCC6803 therefore holds significant promise for CO₂ capture and utilization.

Keywords: Autofermentation; Cyanobacteria; Dynamic metabolic profiling; Metabolomics; Succinate; Synechocystis.

Fig. 1

Time-course of the production of organic acids secreted into the medium and glycogen utilization during dark anoxic fermentation by Synechocystis 6803 phototrophically cultivated in the presence of 5 mM NaNO₃ (open symbols) or 5 mM NH₄Cl (closed symbols) as the sole nitrogen source. Each data point represents the average (±SD) of three independent experiments

Fig. 2

Glycogen phosphorylase activity under light and dark anoxic conditions. The activity was measured after 72 h phototrophic cultivation and 24 h dark anoxic cultivation. Each data point represents the average (±SD) of three independent experiments.

Fig. 3

Time-course changes of intracellular metabolite pool sizes during dark anoxic fermentation by Synechocystis 6803 phototrophically cultivated in the presence of 5 mM NaNO₃ (open symbols) or 5 mM NH₄Cl (closed symbols) as the sole nitrogen source. Two-fold upregulated and downregulated genes are shown in red and blue characters, respectively. A reaction catalyzed by ACL is shown with dashed lines. Abbreviations: FBP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; GABA, γ-aminobutyrate; GAP, glyceraldehyde 3-phosphate; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvate; 2PGA, 2-phosphoglycerate; 3PGA, 3-phosphoglycerate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; S7P, sedoheptulose 7-phosphate; SBP, sedoheptulose 1,7-bisphosphate; DCW, dry cell weight. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Time-course changes in the metabolite ¹³C fraction following the addition of ¹³C-glucose to cultures of Synechocystis sp. PCC6803 phototrophically cultivated in the presence of 5 mM NaNO₃ (open symbols) or 5 mM NH₄Cl (closed symbols) as the sole nitrogen source. Each data point represents the average (±SD) of three independent experiments.

Fig. 5

Organic acid production in Ppc-ox and its vector control strain in the absence or presence of 100 mM NaHCO₃. Organic acids and OD₇₅₀ were measured after 72 h fermentation. Values represent the average (±SD) of three independent experiments.

Fig. 6

Effect of NaHCO₃ addition on organic acid production by Synechocystis sp. PCC6803 GT strain and its effect on glycogen utilization. Organic acids and glycogen were measured after 72 h fermentation. Values represent the average (± SD) of three independent experiments.

Fig. 7

Time-course changes in the metabolite ¹³C fraction following the addition of ¹³C-bicarbonate. Each data point represents the average (±SD) of three independent experiments.