Functional analysis and enzymatic modification by site-directed mutagenesis of an omega-6 fatty acid desaturas from Arctic Chlamydomonas sp.

Abstract

Arctic <em>Chlamydomonas</em> 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 <em>Chlamydomonas</em> 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 FADs, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. Arctic <em>Chlamydmonas</em> sp. omega-6 fatty acid desaturase with 48.2 kDa showed enzymatic activity enhancing the concentration of linoleic fatty acid in the <em>E. coli</em> expression system. 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 A<em>Ch</em>FAD6-transformed cells increased more than three-fold (6.73 ± 0.13 mg g^-1 dry cell weight (DCW)) compared with cells transform- ed with vector alone. In contrast, transformation with Mut-A<em>ChFAD</em>6 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 A<em>Ch</em>FAD6 of Arctic<em> Chlamydomonas</em> sp. increases membrane fluidity by enhancing desaturating C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems. Therefore, genetic modification on the membrane-spanning regions of fatty acid desaturases might increase the yield of target fatty acids and modulate enzymatic activities for industrial application.