Application of label-free shotgun proteomics for the analysis of metabolic pathways in Euglena gracilis and its potential as a source of food supplements

Proteomic studies provide insight into the global expression patterns of proteins within cells. Proteomic profiling of the freshwater protist Euglena gracilis has not been previously investigated, although its transcriptome has recently been published. This work aimed at profiling and identifying proteins involved in carbon metabolism, the biosynthetic and degradation pathways of paramylon, and the biosynthetic pathways of α-tocopherol, ascorbate and the twenty protein-building amino acids under photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivations. Paramylon content was the highest under HT condition, with the streptomycin-bleached mutant E. gracilis ZSB synthesising the most paramylon and the wild-type E. gracilis var. saccharophila synthesising paramylon the fastest. However, antioxidant content was the highest under PT condition, with the wild-type E. gracilis Z strain accumulating the most antioxidants. The abundance of some free amino acids varied between the mid-exponential phase and the beginning of the stationary phase, and between growth conditions, but the total amount remained about the same with arginine as the most abundant amino acid. Label-free shotgun proteomics enabled identification of over 4000 translated proteins, about 30% of which could not be annotated by sequence similarity alone. Many enzymes exhibited several isoforms that were influenced by growth condition. Not all isozymes identified in the transcriptome were detected in the proteome, suggesting post-transcriptional regulation. The results indicated diversity of pathways similar to different organisms, such as lysine biosynthesis to fungi, and serine and proline biosyntheses to plants. Some pathways were unique to Euglena, such as the TCA cycle, and paramylon, ascorbate and arginine biosyntheses. The proteomic studies revealed that instead of hexokinase E. gracilis uses a high-specificity glucokinase for the EMP pathway. Two paramylon synthase candidates (EgGSL1 and EgGSL2) were identified of which EgGSL2 was predominant and expressed under all growth conditions. EgGSL1 was not expressed under HT condition, but the gene transcript was detected by qRT-PCR across all growth conditions indicating light induction and post-transcriptional regulation. Some enzymes of the Calvin pathway were expressed under HT cultivation suggesting post-translational regulation. HT cells may also carry out CO2 fixation in the dark even in the presence of sufficient glucose in the medium. Two pathways for serine biosynthesis were identified, one of which was prevalent under PT and MT cultivation, and the other under HT cultivation. The MT cultivation of E. gracilis var. saccharophila was further chosen for evaluation of this strain as a potential source of food supplements in a laboratory-scale bioreactor, as this strain produced higher paramylon than E. gracilis Z, and under MT cultivation its antioxidant levels were higher than those of E. gracilis ZSB. This work expands on the existing knowledge of metabolic pathways in E. gracilis and provides insight into how these pathways are influenced by growth condition, thus providing a foundation for future strain engineering.