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.
Highlights
Recent research has been performed on the carbon and energy metabolism of C. tepidum [4, 5], rigorous quantification of the metabolic pathway activities has not yet been achieved
This paper reports on the first studies of the fluxomics of mixotrophic metabolism in the green sulfur bacteria and provides complementary information to previous genomic and proteomic studies
The fluxes to biomass pools were loosely constrained by the estimated dry cell weight (DCW) and biomass compositions
Summary
13C-labeled Experiments—C. tepidum cultures were grown anaerobically at temperatures ranging from 46 –50 °C in low intensity light (100 Ϯ 10 micromoles/m2/s). Biomass was sampled at two time points (4-h interval) in the middle exponential growth phase, and the labeling patterns of proteinogenic amino acids in the biomass were measured. The invariability of amino acid labeling during the two time points confirmed the pseudo-steady-state metabolism in tracer experiments. Metabolites and Isotopomer Analysis—The amount of pyruvate and acetate during the growth period was determined by enzymatic assays [21,22,23]. For each type of fragments, the labeling patterns were represented by M0, M1, M2, etc, which were fractions of unlabeled, singly labeled, and doubly labeled amino acids. To compare the relative contributions of carbon substrates and CO2 to mixotrophic biomass synthesis, the substrate utilization ratio R was calculated based on the labeling patterns of unfragmented amino acid X (e.g. alanine) [7],
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