Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum - PubMed (original) (raw)
Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum
Xueyang Feng et al. J Biol Chem. 2010.
Abstract
The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80∼100 units for mixotrophic cultures grown on acetate and 200∼230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.
Figures
FIGURE 1.
Citramalate pathway for isoleucine biosynthesis in C. tepidum (using [2-13C]acetate and NaHCO3 as carbon sources). The asterisks indicate the positions of labeled carbon. The dashed lines indicate inactive pathways.
FIGURE 2.
Metabolic flux distribution in C. tepidum. A, net flux distribution in acetate growth conditions (based on [2-13C]acetate culture). B, net flux distribution in pyruvate growth conditions. The S.D. and exchange coefficients are marked by flux ± S.D. and < exchange coefficient >, respectively. The inactive pathways are marked with dashed lines. The calculated biomass yield (moles of carbon in biomass/mol of substrate): pyruvate culture (YX/S = 9.2); acetate culture (YX/S = 6.4). S7P, sedoheptulose-7-phosphate; SUCC, succinate; SUCCoA, succinyl-CoA; Xu5P, xylulose-5-phosphate; 3PG, 3-phosphoglycerate; 6PG, 6-phosphogluconate; AC, intracellular acetate; ACCOA, acetyl-coenzyme A; AC.ext, extracellular acetate; AKG, α-ketoglutarate; CIT, citrate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose 1,6-bisphosphate; FNR, ferredoxin-NAD(P)+ reductase; FUM, fumarate; G6P, glucose-6-phosphate; GAP, glyceraldehyde 3-phosphate; ICIT, isocitrate; MAL, malate; OAC, oxaloacetate; PEP, phosphoenolpyruvate; PYR, intracellular pyruvate; PYR.ext, extracellular pyruvate; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; PP, pentose phosphate.
FIGURE 3.
Model quality test for acetate metabolism (A) and pyruvate metabolism (B). ●, alanine; ○, serine; ▾, aspartate, Δ, glutamate; ■, leucine; □, histidine; ♢, phenylalanine; and ♦, glycine.
FIGURE 4.
Proposed energy metabolism in C. tepidum. A, energy requirement (mmol/g DCW/h) in acetate growth conditions. B, energy requirement (mmol/g DCW/h) in pyruvate growth conditions. The intracellular energy metabolism was quantified in the framed figures based on the relative flux distributions. (Detailed calculations can be found in
supplemental Table S4, A and B
). Arrows pointing to the framed figure indicated the energy demand of intracellular metabolism. Arrows pointing to biomass indicate the energy demand of biomass accumulation. Arrows pointing from light indicated the entire energy harvested by C. tepidum. The light reaction produces reduced ferredoxin and ATP. NADPH and NADH are mainly generated by ferredoxin-NAD(P)+ reductase. Biomass (protein) synthesis can also generate a small amount of NADH, as indicated in the figure. SUCC, succinate; SUCCoA, succinyl-CoA; 3PG, 3-phosphoglycerate; ACCOA, acetyl-coenzyme A; AC.ext, extracellular acetate; AKG, α-ketoglutarate; CIT, citrate; FNR, ferredoxin-NAD(P)+ reductase; FUM, fumarate; ICIT, isocitrate; MAL, malate; OAC, oxaloacetate; PEP, phosphoenolpyruvate; PYR, intracellular pyruvate; KGOR, 2-ketoglutarate ferredoxin oxidoreductase; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin; PP, pentose phosphate.
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