New insights into {gamma}-aminobutyric acid catabolism: Evidence for {gamma}-hydroxybutyric acid and polyhydroxybutyrate synthesis in Saccharomyces cerevisiae

Abstract

The gamma-aminobutyrate (GABA) shunt, an alternative route for the conversion of alpha-ketoglutarate to succinate, involves the glutamate decarboxylase Gad1p, the GABA transaminase Uga1p and the succinate semialdehyde dehydrogenase Uga2p. This pathway has been extensively described in plants and animals, but its function in yeast remains unclear. We show that the flux through Gad1p is insignificant during fermentation in rich sugar-containing medium, excluding a role for this pathway in redox homeostasis under anaerobic conditions or sugar stress. However, we found that up to 4 g of exogenous GABA/liter was efficiently consumed by yeast. We studied the fate of this consumed GABA. Most was converted into succinate, with a reaction yield of 0.7 mol/mol. We also showed that a large proportion of GABA was stored within cells, indicating a possible role for this molecule in stress tolerance mechanisms or nitrogen storage. Furthermore, based on enzymatic and metabolic evidence, we identified an alternative route for GABA catabolism, involving the reduction of succinate-semialdehyde into gamma-hydroxybutyric acid and the polymerization of gamma-hydroxybutyric acid to form poly-(3-hydroxybutyric acid-co-4-hydroxybutyric acid). This study provides the first demonstration of a native route for the formation of this polymer in yeast. Our findings shed new light on the GABA pathway and open up new opportunities for industrial applications.

FIG. 1.

GABA uptake and catabolism as a function of yeast central carbon metabolism. The following processes are as denoted: GABA uptake (arrow 1); endogenous formation of GABA from α-KG, involving a glutamate dehydrogenase (arrow 2) and a GAD (arrow 3); conversion of GABA into succinate through a GABA transaminase (arrow 4) and an SSA dehydrogenase (arrow 5); formation of succinate by oxidative decarboxylation of α-KG via the α-KG dehydrogenase complex (arrow 6); formation of succinate through the reductive branch of the TCA cycle, involving fumarate reductase (arrow 7); SSA reduction by the GHB dehydrogenase (arrow 8); polymerization of GHB and 3-HB by P(3HB-co-4HB) synthetase (arrow 9); 3HB formation from acetyl-CoA involving an acetoacetyl-CoA thiolase (arrow 10) and a 3HB-CoA dehydrogenase (arrow 11); and vacuolar import of GABA (arrow 12). Metabolic routes and gene names previously identified in yeast are indicated by black lines. Metabolic routes identified in the present study are indicated by gray lines.

FIG. 2.

Expression of GAD1, UGA1, and UGA2 during fermentation of synthetic MS medium by the V5 strain. (A) Growth (circles), assimilable nitrogen (triangles), and GABA (squares) consumption curves for fermentation by V5 in the absence (closed symbols) or presence of 500 mg of GABA/liter (open symbols). The arrow indicates the point at which growth is arrested. The data presented here are representative of three independent experiments. (B) Northern blot analysis of GAD1, UGA1, and UGA2 mRNAs during the course of fermentation on MS in the presence or absence of GABA.

FIG. 3.

Succinate formation during fermentation on the synthetic media MS and MG. (A) Production of succinate by V5 (circles), V5 gad1 (squares), and V5 uga2 (triangles) strains during fermentation in MS (closed symbols) or MG (open symbols). The arrow indicates the time at which growth was arrested. The data presented here are representative of three experiments. (B) Formation of GABA by V5, V5 gad1, and V5 uga2 strains at the end of fermentation in MS (▪) or MG (□). The data are means ± the standard deviation of three independent experiments.

FIG. 4.

Impact of the presence of extracellular GABA on the formation of succinate during fermentation. V5 (circles), V5 gad1 (squares), and V5 uga2 (triangles) strains were grown in MS with (open symbols) or without (closed symbols) 1 g of GABA/liter; succinate production was measured during the course of fermentation. The data presented here are representative of three independent experiments.

FIG. 5.

GABA consumption and conversion to succinate. GABA consumption by the wild-type strain V5 (circles) and the uga2 mutant (triangles) was determined by measuring residual levels of GABA in the culture medium at the end of fermentation, with an initial GABA concentration of 0 to 40 mM. All values are means ± the standard deviations of three independent experiments. For V5 fermentations, the molar yield for the conversion of GABA into succinate by the strain was calculated (dashed line).

FIG. 6.

Intracellular accumulation of GABA. V5 and uga2 cells were harvested after 17 h (growth phase, □) or 44 h (stationary phase, ▪) of fermentation in MS in the presence of 20 mM GABA for determination of the GABA content.