13C-tracer and gas chromatography-mass spectrometry analyses reveal metabolic flux distribution in the oleaginous microalga Chlorella protothecoides

Abstract

The green alga Chlorella protothecoides has received considerable attention because it accumulates neutral triacylglycerols, commonly regarded as an ideal feedstock for biodiesel production. In order to gain a better understanding of its metabolism, tracer experiments with [U-(13)C]/[1-(13)C]glucose were performed with heterotrophic growth of C. protothecoides for identifying the metabolic network topology and estimating intracellular fluxes. Gas chromatography-mass spectrometry analysis tracked the labeling patterns of protein-bound amino acids, revealing a metabolic network consisting of the glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle with inactive glyoxylate shunt. Evidence of phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme activity was also obtained. It was demonstrated that the relative activity of the pentose phosphate pathway to glycolysis under nitrogen-limited environment increased, reflecting excess NADPH requirements for lipid biosynthesis. Although the growth rate and cellular oil content were significantly altered in response to nitrogen limitation, global flux distribution of C. protothecoides remained stable, exhibiting the rigidity of central carbon metabolism. In conclusion, quantitative knowledge on the metabolic flux distribution of oleaginous alga obtained in this study may be of value in designing strategies for metabolic engineering of desirable bioproducts.

Figure 1.

Structural units for biomass formation in C. protothecoides under different nitrogen concentrations. The numbers represent percentages of the corresponding components in biomass (g g⁻¹ dry weight).

Figure 2.

Origin of metabolic intermediates in C. protothecoides during nitrogen-limited (white bars) and nitrogen-sufficient (black bars) growth. Asterisks indicate results obtained from 100% [1-¹³C]Glc labeling experiments. Other results were from 10% U-¹³C-labeled and 90% unlabeled Glc cultures. In certain cases, the MS data permit the determination only of upper bounds (ub) or lower bounds (lb) on the origin of intermediates. Mean relative split ratios ± sd are given for parallel incubations. E4P, Erythrose-4-phosphate; G6P, Glc-6-P; GOX, glyoxylate shunt; Mal, malate; P5P, pentose-5-phosphate; tkt, transketolase.

Figure 3.

Flux maps of central carbon metabolism of C. protothecoides under sufficient (left) and limited (right) nitrogen conditions. The estimated net fluxes were percentages of the relative rates normalized to the Glc uptake rates, which were 0.80 mmol g⁻¹ dry weight h⁻¹ in the nitrogen-sufficient culture and 0.65 mmol g⁻¹ dry weight h⁻¹ in the nitrogen-limited culture. Directions of net fluxes are represented by arrows. The gray arrows indicate flux related to biomass formation. The flux distributions were obtained from the best fit to the quantitative physiological data and the constraints derived from the MS measurements. F6P, Fru-6-P; T3P, triose-3-phosphate; AcCoA, acetyl-CoA; ICT, isocitrate; AKG, α-ketoglutarate; S7P, sedoheptulose-7-phosphate. [See online article for color version of this figure.]

Figure 4.

Flux distribution at several branch points adjacent to fatty acid synthesis. The width of arrows is proportional to the flux of carbon through a particular step calculated from the flux estimates. The top and bottom numbers represent relative flux during nitrogen-sufficient and nitrogen-limited cultivation, respectively. [See online article for color version of this figure.]