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

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Title
Functional analysis and enzymatic modification by site-directed mutagenesis of an omega-6 fatty acid desaturas from Arctic Chlamydomonas sp.
Other Titles
북극 클라미노모나스 유래 오메가-6 지방산 불포화 효소의 기능 분석 및 위치 특이적 돌연변이에 의한 효소활성 변화 연구
Authors
Jung, Woongsic
Kim, Sanghee
Choi, Han-Gu
Kang, Sung-Ho
Han, Se Jong
Kim, Eun Jae
Keywords
북극; 불포화지방산효소; 클라미도모나스
Issue Date
2016
Citation
Jung, Woongsic, et al. 2016. Functional analysis and enzymatic modification by site-directed mutagenesis of an omega-6 fatty acid desaturas from Arctic Chlamydomonas sp.. 제30회 한국조류학회 학술발표대회 및 국제 심포지엄. 제주 라마다프라자 제주호텔. 2016.09.28~2016.09.30.
Abstract
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 FADs, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. 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) 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 transform- ed 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 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.
URI
http://repository.kopri.re.kr/handle/201206/7168
Conference Name
제30회 한국조류학회 학술발표대회 및 국제 심포지엄
Conference Place
제주 라마다프라자 제주호텔
Conference Date
2016.09.28~2016.09.30
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