Optimization of energy production and central carbon metabolism in a non-respiring eukaryote

Abstract

Most eukaryotes respire oxygen, using it to generate biomass and energy. However, a few organisms have lost the capacity to respire. Understanding how they manage biomass and energy production may illuminate the critical points at which respiration feeds into central carbon metabolism and explain possible routes to its optimization. Here, we use two related fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces japonicus, as a comparative model system. We show that although S. japonicus does not respire oxygen, unlike S. pombe, it is capable of efficient NADH oxidation, amino acid synthesis, and ATP generation. We probe possible optimization strategies through the use of stable isotope tracing metabolomics, mass isotopologue distribution analysis, genetics, and physiological experiments. S. japonicus appears to have optimized cytosolic NADH oxidation via glycerol-3-phosphate synthesis. It runs a fully bifurcated TCA pathway, sustaining amino acid production. Finally, we propose that it has optimized glycolysis to maintain high ATP/ADP ratio, in part by using the pentose phosphate pathway as a glycolytic shunt, reducing allosteric inhibition of glycolysis and supporting biomass generation. By comparing two related organisms with vastly different metabolic strategies, our work highlights the versatility and plasticity of central carbon metabolism in eukaryotes, illuminating critical adaptations supporting the preferential use of glycolysis over oxidative phosphorylation.

Keywords: Schizosaccharomyces japonicus; Schizosaccharomyces pombe; TCA Cycle; bifurcated TCA pathway; fermentation; glycolysis; metabolomics; respiration.

Figure 1. S. japonicus does not respire but oxidizes NADH efficiently

(A) Oxygen consumption rates of S. pombe and S. japonicus wild-type (WT) and cox6⊿ cultures in YES medium. Means are derived from three biological replicates. (B) Growth rates of indicated strains in EMM medium. Means are derived from at least five biological repeats with three technical replicates. (C) Cellular NAD⁺/NADH ratios. Means are derived from at least four biological replicates. (D) Production of ¹³C-labeled glycerol-3-phosphate after 1 min of ¹³C₆-glucose exposure. Means represent two biological and two technical replicates. (A–D) Error bars represent ±SEM; p values are derived from unpaired t test. (E and F) Growth curves of S. pombe strains (E) in EMM medium and S. japonicus strains (F) in YES medium. Error bars represent ±SD. Shown are the means of OD₅₉₅ readings derived from three technical replicates, representative of three biological repeats. See also Figure S1 and Data S1A.

Figure 2. S. japonicus operates a bifurcated TCA pathway and efficiently synthesizes TCA-derived amino acids

(A) Isotopologs of intermediates expected in the oxidative TCA cycle, after feeding ¹³C₆-glucose. Pink arrow: the reaction catalyzed by succinate dehydrogenase (SDH). Isotopologs in green originate from the first cycle (M + 2 acetyl-CoA and M + 0 oxaloacetate). Isotopologs in blue are expected to be generated after the 4^th cycle.^,– (B) Isotopologs originating from the bifurcated TCA pathway. M + 3 pyruvate may be carboxylated using M + 0 CO₂, leading to M + 3 oxaloacetate/aspartate (red),^,– or M + 1 CO₂, leading to M + 4 oxaloacetate/aspartate (orange). (C–E) M + 2 (C), M + 3 (D), and M + 4 (E) fumarate fractions relative to the entire fumarate pool 30 min after ¹³C₆-glucose addition. (F and G) ¹³C-labeled alpha-ketoglutarate (F) and glutamate (G) fractions 30 min after ¹³C₆-glucose addition. (C–G) Shown are mean ±SEM of two biological and two technical replicates, p values were calculated using unpaired t test. (H) Growth rates of S. pombe and S. japonicus grown in EMM with 0.2 g/L of either glutamate (E), glutamine (Q), or arginine (R). Mean ±SEM of three technical and at least two biological replicates are shown, with p values generated using unpaired t test. See also Figure S2 and Data S1D.

Figure 3. S. japonicus maintains higher glycolytic activity than S. pombe

(A) Glycolysis and its outputs. Blue: plasma membrane hexose transporter. Underlined: metabolites quantified in this study. Pyruvate kinase Pyk1 is indicated, with A and T denoting the point mutation at site 343. (B) Whole-cell ATP/ADP ratios. Mean ±SEM values of at least four biological replicates. p values were calculated using unpaired t test. (C) Rate of glucose uptake in EMM. Mean ± SEM of at least five biological replicates are shown. p values were calculated using unpaired t test. (D)3-phosphoglycerate abundance relative to the sum of detected glycolytic intermediates (G6P, F6P, DHAP, 3PGA, 2PGA, PEP, and pyruvate). (E) Glucose-6-phosphate levels relative to fructose-6-phosphate. (D and E) Shown are mean ±SEM of three technical and two biological replicates. p values were calculated using unpaired t test. See also Figure S3 and Data S1E.

Figure 4. S. japonicus may upregulate the entry into the pentose phosphate pathway

(A) PPP and its intersection with glycolysis. Bold: metabolites quantified in this study. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; Phe, phenylalanine; Tyr, tyrosine. (B) Glucose-6-phosphate abundance normalized to 6-phosphogluconate. (C) Sum of oxidative PPP intermediates (6PGA and Ru5P) relative to non-oxidative PPP intermediates (R5P and SH7P). (B and C) Dotted lines indicate the ratio of 1. (D)¹³C-labeled phenylalanine and tyrosine fractions 10 min after ¹³C₆-labeled glucose addition. (B–D) Mean ±SEM of two biological and two to three technical replicates. Statistical analyses were performed using unpaired t test. (E) Cellular NADPH relative to a total NADP(H) pool. Mean ±SEM of three biological replicates, p values estimated using unpaired t test. (F) A diagram summarizing the findings of this study. ETC, electron transport chain; OXPHOS, oxidative phosphorylation. See also Figure S4 and Data S1E–S1G.