Genome-based Primary Metabolic Network Reconstruction And ¹³C Flux Analysis Of The Oleaginous Microalga Chlorella Protothecoides

The green alga Chlorella protothecoides capable of autotrophic and heterotrophic growth with rapid growth and lipid accumulation is a promising candidate for biofuel production. So far, few are known about the mechanism leading to the phenotypic differences under various nutritional conditions. In this work, we reconstructed the primary metabolic network, and combined metabolomics with 13 C flux analysis to systematically investigat the metabolism of C. protothecoides.We first established a metabolite extraction approach and compound detection method based on LC-MS, and compared the metabolomes of C. protothecoides under autotrophic and heterotrophic conditions. Variations are present in the majority of metabolites, especially for those involved in sugar catabolism. Intracellular concentration of ATP and reducing equivalents, as well as acetyl-CoA and malonyl-CoA presented great increase in heterotrophic growth, indicating that glucose-fed condition favors energy-intensive metabolism such as fatty acid biosynthesis. According to metabolic profiling of the transition from autotrophic to heterotrophic mode, most free amino acids decreased during the metabolic shift, while intermediates in glycolysis, the pentose phosphate pathway and tricarboxylic acid cycle increased dramatically, which suggests that protein accumulated in autotrophic phase was recycled, and the carbon skeleton is channeled to fatty acid synthesis.Based on the newly sequenced genome, we reconstructed the primary metabolic network of C. protothecoides, and simulated the growth under different conditions. The simulated results matched well with the experimentally determined ones, which validated the model and data consistency. According to optimal carbon utilization, the potential of autotrophic and heterotrophic growth rate which could be further improved 20% and 10%, respectively. Phenotype phase plane analysis showed that the optimal growth and lipid accumulation appears when the ratio of photon uptake to CO2 consumption rate is around 8.4 for autotrophic cells, and the ratio of glucose uptake to O2 consumption is 2.7 for heterotrophic cells.We applied isotopically nonstationary metabolic flux analysis to our metabolic network, tracing the kinetics of isotope labeling trajectories and quantifying the fluxes in C. protothecoides. The result reveals negligible photorespiratory fluxes and metabolically less active TCA cycle in phototrophic cells, which might function in biosynthesis instead of generating energy. In heterotrophic C. protothecoides, high activity of C4 pathway was observed, presumably supplying reducing equivalents. Inhibition of citrate synthase is a feasible strategy to optimize the flux to fatty acid synthesis. Meanwhile, alternative pathways generating the reducing equivalents are desired.Kinetic flux analysis applied in the investigation of nitrogen metabolism revealed that glutamate dehydrogenase is the main route for nitrogen assimilation in autotrophic C. protothecoides, while the glutamine synthetase-glutamate synthase cycle is responsible for the ammonia uptake in heretotrophically grown cells. Choice of different nitrogen assimilation pathway by C. protothecoides demonstrated its ability to self-adjust to various environmental stresses.These integrated studies of metabolic network coupled with metabolomics and flux analysis shed light on the metabolism of oleaginous microalga C. protothecoides and may serve as a cutting-edge toolbox for the systematic engineering of microalgae for biofuel production.