Simple, effective protein extraction method and proteomics analysis from polyunsaturated fatty acids-producing micro-organisms.

Polyunsaturated fatty acids (PUFAs) are valuable ingredients in the food and pharmaceutical products due to their beneficial influence on human health. Most studies paid attention on the production of PUFAs from oleaginous micro-organisms but seldom on the comparative proteomics of cells. In the study, three methods (i.e., cold shock, acetone precipitation and ethanol precipitation) for lipid removal from crude protein extracts were applied in different PUFAs-producing micro-organisms. Among the selective strains, Schizochytrium was used as an oleaginous strain with high lipid of 60.3 (w/w%) in biomass. The Mortierella alpina and Cunninghamella echinulata were chosen as the low-lipid-content strains with 25.8 (w/w%) and 21.8 (w/w%) of lipid in biomass, respectively. The cold shock resulted as the most effective method for lipid removed, thus obtained higher protein amount for Schizochytrium. Moreover, from the comparative proteomics for the three PUFAs-producing strains, it showed more significant proteins of up or down-regulation were explored under cold shock treatment. Therefore, the essential proteins (i.e., polyunsaturated fatty acid synthase) and regulating proteins were observed. In conclusion, this study provides a valuable and practical approach for analysis of high PUFAs-producing strains at the proteomics level, and would further accelerate the understanding of the metabolic flux in oleaginous micro-organisms.

Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production.

Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.