Genetic isolation and functional analysis of stearoyl-CoA desaturase and omega-6 fatty acid desaturase from polar psychrophilic microorganisms

Abstract

Stearoyl-CoA desaturase (SCD, Δ9 desaturases) is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two SCD genes were discovered from an Antarctic copepod, Tigriopus kingsejongensis, which were collected in a tidal pool near the King Sejong Station, Antarctica. The molecular weights deduced from both genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various types of organisms and used to analyze the relationships among TkSCDs. The enzymatic functions of both SCDs, as assessed by heterologous expression of recombinant proteins in E. coli, revealed that the amount of C16:1 and C18:1 fatty acids increased by more than three-fold after induction with isopropyl β-D-thiogalactopyranoside. Especially, 19.36 ± 1.34% and 14.04 ± 1.41% of C18:1 fatty acids were produced in cells expressing TkSCD-1 and TkSCD-2, respectively. The results of this study suggest that both TkSCD genes encode a functional desaturase that is capable of increasing the amounts of palmitoleic fatty acids and oleic fatty acids in a prokaryotic expression system. 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. 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. Arctic Chlamydmonas sp. omega-6 fatty acid desaturase with 48.2 kDa showed enzymatic activity enhancing the concentration of linoleic fatty acid in the E. coli expression system. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) proteins. Quantitative analysis 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) 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 SCD. These results demonstrate that AChFAD6 increases membrane fluidity by enhancing desaturating C18 fatty acids in prokaryotic systems. In addition, mutation on the membrane-spanning regions of fatty acid desaturases might increase the yield of target fatty acids and modulate enzymatic activities for industrial application.