Isolation of enhanced eicosapentaenoic acid producing mutants of Nannochloropsis oculata ST-6 using ethyl methane sulfonate induced mutagenesis techniques and their characterization at mRNA transcript level

Ethyl methane sulfonate-induced mutagenesis techniques were used to obtain cerulenin and erythromycin resistant mutant strains, which show a marked increase of 29 and 12%, respectively, in eicosapentaenoic acid content compared to the wild type (WT) strain. The increase in eicosapentaenoic acid content in both strains correlated well with a concomitant increase in 14:0, 16:0, 16:1, 18:0 and 20:4 n - 6 content of fatty acids. However, the linoleic acid content increased only in the cerulenin resistant strain, whereas the erythromycin resistant strain was completely devoid of this fatty acid, providing a contrasting feature for further study. Partial characterization of the mutant strains based on whole cell ultrastructure, growth patterns with or without selection pressure as well as at different temperatures revealed that the mutants were more thermotolerant, and maintained their morphological and genomic fidelity, although minor changes in band patterns were noted with random amplified polymorphic DNA techniques compared to the WT strain. Therefore, the developed new strains could have wider applications as planktonic feed for the mariculture industry. Further characterization of the mutants at the molecular level revealed that steady-state mRNA transcript levels of chloroplast-localized Delta 12 desaturase and stearoyl-ACP desaturase genes showed a contrasting transcript profile. However, the level of endoplasmic reticulum-localized Delta 12 desaturase mRNA transcript in either of the mutant strains was almost equal to that of the WT strain. Based on this observation, we postulate that the complete loss of 18:2 fatty acid content in the erythromycin resistant strain might be a result of mutation in the chloroplast-localized Delta 12 desaturase gene and that the increased content of very long chain-polyunsaturated fatty acids might result from extrachloroplastic lipid desaturase activity, a phenomenon similar to that in the majority of angiosperms.