Disruption of cyanobacterial γ-aminobutyric acid shunt pathway reduces metabolites levels in tricarboxylic acid cycle, but enhances pyruvate and poly(3-hydroxybutyrate) accumulation

Abstract

The photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 assimilates carbon dioxide as the sole carbon source, and a major portion of the assimilated carbon is metabolically consumed by the tricarboxylic acid (TCA) cycle. Effects of partial interference of TCA cycle metabolic activity on other carbon metabolism have yet to be examined. Here, the γ-aminobutyric acid (GABA) shunt, one of the metabolic pathways for completing TCA cycle in Synechocystis, was disrupted via inactivating the glutamate decarboxylase gene (gdc). Under normal photoautotrophic condition, cell growth and the level of the TCA cycle metabolites succinate, malate and citrate were decreased by 25%, 35%, 19% and 28%, respectively, in Δgdc mutant relative to those in the wild type (WT). The cellular levels of glycogen and total lipids of the Δgdc mutant were comparable to those of the WT, but the intracellular levels of pyruvate and bioplastic poly(3-hydroxybutyrate) (PHB) were 1.23- and 2.50-fold higher, respectively, in Δgdc mutant. Thus, disruption of the GABA shunt pathway reduced the TCA cycle metabolites levels, but positively enhanced the bioaccumulation of pyruvate and PHB. The PHB production rate in Δgdc mutant was 2.0-fold higher than in the WT under normal photoautotrophy.

Figure 1

PHB accumulation and biomass levels under normal photoautotrophic condition. Log-growth-phase cells of the wild type (WT) and Δgdc mutant were diluted and grown in the presence or absence of glutamate (Glu) at 0–200 mM. Data are shown as the mean ± 1 SD derived from three to six independent cultures.

Figure 2

Metabolites levels and enzymatic activities of the Δgdc mutant as relative values to those of the wild type (shown in rectangle boxes). Log-growth-phase cells were diluted and cultured under the normal photoautotrophic condition (0 mM Glu) for 21 d. Metabolites, direct reactions (line arrows), abbreviated pathways (broken arrows) and enzymes (italic capital letters: GABA-AT, γ-aminobutyrate aminotransferase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; OGDC, 2-oxoglutarate decarboxylase; PHAS, polyhydroxyalkanoate (PHA) synthase; SSADH, succinic semialdehyde dehydrogenase) are illustrated. The 2-oxoglutarate/succinyl semialdehyde/succinate route (Oxo/Sucsem/Suc route, shown by purple arrows) and GABA shunt route (green arrows) for completing the Synechocystis TCA cycle are indicated. Data are obtained from four to six independent cultures. Asterisks indicate the ratios for which the levels in Δgdc were significantly different from those in the WT (*P < 0.05; **P < 0.01: unpaired two-tailed t-test). This metabolic connection section was redrawn from the reported metabolic network of Synechocystis sp. PCC6803^–,. The values of metabolites levels and enzymatic activities of the wild type (WT) and Δgdc mutant are given in Table S1, Supplementary Information.

Figure 3

PHB and biomass levels under nitrogen deprivation (-N) or phosphorus deprivation (-P). Log-growth-phase cells of the wild type (WT) and Δgdc mutant were diluted and cultured in either -N or -P medium in the presence of glutamate (Glu) at 0, 10 or 50 mM. Data are shown as the mean ± 1 SD derived from three or four independent cultures.