Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

Abstract

For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that N-acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gadA gene from Synechocystis sp. PCC 6803 was heterologously expressed in Synechococcus sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven Synechococcus sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in Synechococcus sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.

Keywords: 2-oxoglutaric acid; GABA shunt; Synechococcus sp. PCC 7002; Synechocystis sp. PCC 6803; TCA cycle; cyanobacteria; photosynthesis; succinic acid semialdehyde.

FIGURE 1

Scheme showing the TCA cycle, the glyoxylate cycle and the GABA shunt. 2-OG, 2-oxoglutarate; SSA, succinic semialdehyde; GABA, γ-aminobutyric acid; ArgD, GABA aminotransferase; GadA, glutamate decarboxylase; GdhA, glutamate dehydrogenase; 2-OGDC/OgdA, 2-oxoglutarate decarboxylase; SSADH/SsaD, succinic semialdehyde dehydrogenase.

FIGURE 2

LC–MS method to determine intracellular SSA contents. (A) Comparison of chromatograms of WT7002 (black line) and 2.5 mM SSA standard (gray line). Black arrow indicates the peak of SSA. (B) Standard curve of SSA within the concentration range of 0 to 1.0 mM, R² = 0.99.

FIGURE 3

Quantification of intracellular SSA. (A)¹³C-labeling kinetics of SSA under photomixotrophic conditions. The dashed line indicates the time point when the culture was switched from light to dark. (B) Intracellular SSA contents of WT7002 and strains SZ001, and SZ002 under photoautotrophic and dark aerobic conditions. The results are mean values of three biological replicates and error bars represent the standard deviations.

FIGURE 4

Metabolites profiling of TCA cycle metabolites in mutant strains. Category “block side” refers to the side of the TCA cycle (reductive side or oxidative side) that hypothetically prevent the synthesis of succinate due to the corresponding mutation(s). Gray bars indicate the fold changes of these metabolites compared to WT7002 levels.

FIGURE 5

Characterizations of purified recombinant proteins. (A) Purified recombinant proteins of the glutamate decarboxylase (GadA), and the N-acetylornithine aminotransferase from both Synechocystis sp. PCC 6803 (ArgD₆₈₀₃) and Synechococcus sp. PCC 7002 (ArgD₇₀₀₂) were analyzed. Left lanes for each enzyme were stained with Coomassie blue and right lanes were detected by immunoblotting with antibodies to the poly-[His]₁₀-tag. (B) HPLC analysis showing the conversion of glutamate to GABA, catalyzed by the purified glutamate decarboxylase (GAD_reaction). (C) HPLC analysis showing the formation of glutamate from L-ornithine catalyzed by the purified N-acetylornithine aminotransferase from both Synechocystis sp. PCC 6803 (ArgD_6803) and Synechococcus sp. PCC 7002 (ArgD_7002). (D) HPLC analysis showing the formation of glutamate from GABA catalyzed by the purified N-acetylornithine aminotransferase from both Synechocystis sp. PCC 6803 (ArgD_6803) and Synechococcus sp. PCC 7002 (ArgD_7002), demonstrating that N-acetylornithine aminotransferase can also function as GABA aminotransferase. Insets in (C,D) represent the enlarged parts of the elution curves from 8 to 10 min to illustrate the changes of the glutamate peaks observed more clearly. It should be noted that the large differences in peak heights occur because the different compounds have very different molar extinction coefficients at the detection wavelength. Control experiments in all of these assays were performed the same way without recombinant proteins added.

FIGURE 6

Metabolites profiling of GABA shunt mutant strains. Relative metabolites concentrations in strains SZ008 and SZ009, which had been grown under photoautotrophic conditions (A) or dark aerobic conditions (B). Relative concentrations for each metabolite in wild-type Synechococcus 7002 (WT7002) under these growth conditions were set to 1 unit for comparison.