Abstract
Integrated and genome-based flux balance analysis, metabolomics, and (13)C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary (13)C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and (13)C labeling better our understanding on global metabolism of oleaginous alga, paving the way to the systematic engineering of the microalga for biofuel production.
Highlights
Integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel
We reconstructed the primary metabolic network of C. protothecoides on the basis of genome information, focusing on central metabolism comprised by the Calvin-Benson cycle, glycolysis, the PP pathway, the tricarboxylic acid cycle, and the biosynthetic pathways of biomass building blocks
The application of flux balance analysis (FBA) led to several conclusions: (1) completeness of metabolic functionality mined in the genome information, (2) achievement of suboptimal growth by autotrophic and heterotrophic cells, and (3) optimality of metabolic parameter for cell growth and fatty acid production
Summary
Integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The metabolic network modeling assisted by experimental metabolomics and 13C labeling better our understanding on global metabolism of oleaginous alga, paving the way to the systematic engineering of the microalga for biofuel production. Biogenesis of neutral lipids was found in Chlorella spp. cells undergoing glucose bleaching, in which the growth is switched from photoautotrophic to heterotrophic mode and accompanied by chlorophyll degradation. This metabolic transition has been incorporated into a highly efficient biofuel production process (Xiong et al, 2010a), whereas many global changes in metabolism, such as degeneration of the chloroplast, redistribution of carbon flux, and. The studies presented here will detail the Chlorella spp. metabolism and pave the way to biofuel production from this microalga with the predictive ability of a constructed metabolic model
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