Enzymatic modification by point mutation and functional analysis of an omega-6 fatty acid desaturase from arctic Chlamydomonas sp.

• Main Authors: Woongsic Jung, Kim, Eun Jae, Han, Se Jong, Kang, Sung-Ho, Han-Gu Choi, Sanghee Kim
• Format: Article in Journal/Newspaper
• Language: unknown
• Published: Figshare 2016
• Subjects: Biophysics; Biochemistry; Medicine; Microbiology; FOS Biological sciences; Cell Biology; Genetics; 39999 Chemical Sciences not elsewhere classified; FOS Chemical sciences; 69999 Biological Sciences not elsewhere classified; Plant Biology; Arctic
• Online Access: https://dx.doi.org/10.6084/m9.figshare.c.3255928.v1; https://figshare.com/collections/Enzymatic_modification_by_point_mutation_and_functional_analysis_of_an_omega-6_fatty_acid_desaturase_from_arctic_i_Chlamydomonas_i_sp_/3255928/1

Arctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI Culture Collection of Polar Microorganisms (KCCPM). Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases (FADs), and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6 -transformed cells increased more than three-fold (6.73 ± 0.13 mg g −1 dry cell weight (DCW)) compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g −1 DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase (SCD). These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.