高山被孢霉Δ6Ⅱ-脱饱和酶的克隆、表达和功能鉴定
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摘要
产油微生物高山被孢霉中Δ6脱饱和酶是决定ω3/ω6脂肪酸代谢流的关键酶,该酶对不同底物的催化特性直接决定该菌体内脂肪酸的流向。在已完成基因组测序的高山被孢霉ATCC32222中经注释发现它存在两种Δ6脱饱和酶(Δ6-Ⅰ和Δ6-Ⅱ),选择对其中的Δ6-Ⅱ脱饱和酶进行克隆、表达和功能鉴定。首先以p YES2/NT C质粒为骨架构建了Δ6-Ⅱ脱饱和酶基因(FADS6-Ⅱ)的表达载体(p YES2/NT C-FADS6-Ⅱ),并转化至酿酒酵母中进行诱导表达,进一步通过在重组菌培养基中添加Δ6脱饱和酶的底物来考察Δ6-Ⅱ脱饱和酶对各底物的偏好作用。实验结果表明,在分别添加0.5 mmol/L亚油酸(LA)和0.5 mmol/Lα-亚麻酸(ALA)时,Δ6-Ⅱ脱饱和酶对LA的转化率为42.94%,对ALA没有催化作用。而当底物添加方式改为同时添加LA和ALA时(分别为0.25 mmol/L),Δ6-Ⅱ脱饱和酶对LA的转化率为37.12%,对ALA仍没有催化作用。该实验结果为高山被孢霉中Δ6-Ⅱ脱饱和酶的催化功能研究提供了理论依据。
Δ6 desaturase from oleaginous Mortierella alpina( Ma FADS6) is a key enzyme in biosynthesis ofω3 /ω6 PUFAs,whose substrate preference directly determines the fatty acid metabolic flux. There are two isozymes: FADS6-Ⅰ and FADS6-Ⅱ in the genome of Mortierella alpina ATCC 32222. In most species,FADS6-Ⅰ has been widely researched,but FADS6-Ⅱ is rarely reported. The substrate preference of Δ6-Ⅱ FADS6 was analyzed. The heterologous expression vector for FADS6-Ⅱ was constructed under the skeleton of plasmid p YES2 /NT C and expressed in Saccharomyces cerevisiae. By adding exogenous substrate,the preference of Δ 6Ⅱ FADS6 was acquired. The results showed that when a single substrate was added( 0. 5 mmol / L linoleic acid [LA,18∶2~(Δ9,12)]or 0. 5 mmol/L α-linolenic acid [ALA,18∶ 3~(Δ9,12,15)]),respectively,only LA can be converted by Δ6-Ⅱ FADS6 and its conversion rate was 42. 94%. When LA and ALA were added at the same time( 0. 25 mmol / L LA+ 0. 25 mmol / L ALA),37. 12% LA was converted whereas no product( stearidonic acid [18: 4~(Δ6,9,12,15)],SDA) from ALA was determined. It will provide theoretical evidence for the function of Δ6-Ⅱ desaturase in M.alpina.
Δ6 desaturase from oleaginous Mortierella alpina( Ma FADS6) is a key enzyme in biosynthesis ofω3 /ω6 PUFAs,whose substrate preference directly determines the fatty acid metabolic flux. There are two isozymes: FADS6-Ⅰ and FADS6-Ⅱ in the genome of Mortierella alpina ATCC 32222. In most species,FADS6-Ⅰ has been widely researched,but FADS6-Ⅱ is rarely reported. The substrate preference of Δ6-Ⅱ FADS6 was analyzed. The heterologous expression vector for FADS6-Ⅱ was constructed under the skeleton of plasmid p YES2 /NT C and expressed in Saccharomyces cerevisiae. By adding exogenous substrate,the preference of Δ 6Ⅱ FADS6 was acquired. The results showed that when a single substrate was added( 0. 5 mmol / L linoleic acid [LA,18∶2~(Δ9,12)]or 0. 5 mmol/L α-linolenic acid [ALA,18∶ 3~(Δ9,12,15)]),respectively,only LA can be converted by Δ6-Ⅱ FADS6 and its conversion rate was 42. 94%. When LA and ALA were added at the same time( 0. 25 mmol / L LA+ 0. 25 mmol / L ALA),37. 12% LA was converted whereas no product( stearidonic acid [18: 4~(Δ6,9,12,15)],SDA) from ALA was determined. It will provide theoretical evidence for the function of Δ6-Ⅱ desaturase in M.alpina.
引文
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[13]代鹏,陈海琴,顾震南,等.高山被孢霉生产多不饱和脂肪酸发酵条件的研究进展.食品工业科技,2014,35(5):354-359.Dai P,Chen H Q,Gu Z N.Research progress in fermentation condition for polyunsaturated fatty acids by Mortierella Alpina.Science and Technology of Food Industry,2014,35(5):354-359.
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[15]Zhou X R,Robert S,Singh S,et al.Heterologous production of GLA and SDA by expression of an Echium plantagineum delta 6-desaturase gene.Plant Science,2007,172(2):421-422.
[16]丁兆坤,麻艳群,许友卿.合成高度不饱和脂肪酸去饱和酶的分子生物学研究II.克隆,表达与功能分析.中国生物工程杂志,2008,28:196-200.Ding Z K,Ma Y Q,Xu Y Q.Studies on molecular biology of the desaturases of highly unsaturated fatty acid biosynthesis-II.Cloning,expression and function analysis.China Biotechnology,2008,28:196-200.
[17]Wang L,Chen W,Feng Y,et al.Genome characterization of the oleaginous fungus Mortierella alpina.Plo S one,2011,6(12):e28319.
[18]Hsiao T Y,Holmes B,Blanch H W.Identification and functional analysis of a delta-6 desaturase from the marine microalga Glossomastix chrysoplasta.Marine Biotechnology,2007,9(2):154-165.
[19]Iskandarov U,Khozin-Goldberg I,Cohen Z.Identification and characterization ofΔ12,Δ6,andΔ5 desaturases from the green microalga Parietochloris incisa.Lipids,2010,45(6):519-530.
[20]Kajikawa M,Yamato K T,Kohzu Y,et al.Isolation and characterization ofΔ6-desaturase,an ELO-like enzyme andΔ5-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris.Plant Molecular Biology,2004,54(3):335-352.
[21]Kim S H,Kim J B,Kim S Y,et al.Functional characterization of a delta 6-desaturase gene from the black seabream(Acanthopagrus schlegeli).Biotechnology Letters,2011,33(6):1185-1193.
[22]Ma X,Yu J,Zhu B,et al.Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata.Chinese Journal of Oceanology and Limnology,2011,29:290-296.
[23]Zahringer U,Domergue F,Abbadi A,et al.In vivo characterization of the first acyl-Co A delta6-desaturase from a member of the plant kingdom,the microalga Ostreococcus tauri.Biochem J,2005,389:483-490.
[24]奥斯伯.精编分子生物学实验指南.第5版.北京:科学出版社,2008.545-581.Ausubel F.Short Protocols in Molecular Biology.Fifth ed.Beijing:Science Press,2008.545-581.
[25]Zhang B,Rong C,Chen H,et al.De novo synthesis of trans-10,cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia lipolytica.Microb Cell Fact,2012,11(1):51.
[2]李会珍,张志军.植物合成长链多不饱和脂肪酸研究进展.中国生物工程杂志,2008,28(12):112-115.Li H Z,Zhang Z J.Progresses on biosynthesis of long chain polyunsaturated fatty acids in plant.China Biotechnology,2008.28(12):112-115.
[3]Hong H,Datla N,Reed D W,et al.High-level production ofγ-linolenic acid in Brassica juncea using aΔ6 desaturase from Pythium irregulare.Plant Physiology,2002,129(1):354-362.
[4]Liu J,Li D,Yin Y,et al.Δ6-Desaturase from Mortierella alpina:c DNA cloning,expression,and phylogenetic analysis.Biotechnology Letters,2011,33(10):1985-1991.
[5]黎志勇,纪晓俊,丛蕾蕾,等.发酵法生产亚麻酸的研究进展.中国生物工程杂志,2010,30:110-117.Li Z Y,Ji X J,Cong L L,et al.Progresses in fermentative production ofγ-linolenic acid.China Biotechnology,2010,30:110-117.
[6]Xue Z,Sharpe P L,Hong S P,et al.Production of omega-3eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica.Nature Biotechnology,2013,31(8):734-740.
[7]Grima E M,Medina A R,Giménez A G,et al.Gram-scale purification of eicosapentaenoic acid(EPA,20:5n-3)from wet Phaeodactylum tricornutum UTEX 640 biomass.Journal of Applied Phycology,1996,8(4-5):359-367.
[8]Liu Y,Tang J,Li J J,et al.Efficient production of triacylglycerols rich in docosahexaenoic acid(DHA)by osmoheterotrophic marine protists.Applied Microbiology and Biotechnology,2014,98(23):9643-9652.
[9]Hao G,Chen H,Du K,et al.Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina.Biotechnology Letters,2014,36(9):1827-1834.
[10]Sakuradani E,Ando A,Shimizu S,et al.Metabolic engineering for the production of polyunsaturated fatty acids by oleaginous fungus Mortierella alpina 1S-4.Journal of Bioscience and Bioengineering,2013,116(4):417-422.
[11]Sakuradani E,Kobayashi Mshimizu S.Δ6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus:Gene cloning and its heterologous expression in a fungus,Aspergillus.Gene,1999,238(2):445-453.
[12]Sakuradani E,Shimizu S.Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acidproducing Mortierella fungus.Bioscience,Biotechnology,and Biochemistry,2003,67(4):704-711.
[13]代鹏,陈海琴,顾震南,等.高山被孢霉生产多不饱和脂肪酸发酵条件的研究进展.食品工业科技,2014,35(5):354-359.Dai P,Chen H Q,Gu Z N.Research progress in fermentation condition for polyunsaturated fatty acids by Mortierella Alpina.Science and Technology of Food Industry,2014,35(5):354-359.
[14]Zhang Q,Li M,Ma H,et al.Identification and characterization of a novelΔ6-fatty acid desaturase gene from Rhizopus arrhizus.FEBS Letters,2004,556(1):81-85.
[15]Zhou X R,Robert S,Singh S,et al.Heterologous production of GLA and SDA by expression of an Echium plantagineum delta 6-desaturase gene.Plant Science,2007,172(2):421-422.
[16]丁兆坤,麻艳群,许友卿.合成高度不饱和脂肪酸去饱和酶的分子生物学研究II.克隆,表达与功能分析.中国生物工程杂志,2008,28:196-200.Ding Z K,Ma Y Q,Xu Y Q.Studies on molecular biology of the desaturases of highly unsaturated fatty acid biosynthesis-II.Cloning,expression and function analysis.China Biotechnology,2008,28:196-200.
[17]Wang L,Chen W,Feng Y,et al.Genome characterization of the oleaginous fungus Mortierella alpina.Plo S one,2011,6(12):e28319.
[18]Hsiao T Y,Holmes B,Blanch H W.Identification and functional analysis of a delta-6 desaturase from the marine microalga Glossomastix chrysoplasta.Marine Biotechnology,2007,9(2):154-165.
[19]Iskandarov U,Khozin-Goldberg I,Cohen Z.Identification and characterization ofΔ12,Δ6,andΔ5 desaturases from the green microalga Parietochloris incisa.Lipids,2010,45(6):519-530.
[20]Kajikawa M,Yamato K T,Kohzu Y,et al.Isolation and characterization ofΔ6-desaturase,an ELO-like enzyme andΔ5-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris.Plant Molecular Biology,2004,54(3):335-352.
[21]Kim S H,Kim J B,Kim S Y,et al.Functional characterization of a delta 6-desaturase gene from the black seabream(Acanthopagrus schlegeli).Biotechnology Letters,2011,33(6):1185-1193.
[22]Ma X,Yu J,Zhu B,et al.Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata.Chinese Journal of Oceanology and Limnology,2011,29:290-296.
[23]Zahringer U,Domergue F,Abbadi A,et al.In vivo characterization of the first acyl-Co A delta6-desaturase from a member of the plant kingdom,the microalga Ostreococcus tauri.Biochem J,2005,389:483-490.
[24]奥斯伯.精编分子生物学实验指南.第5版.北京:科学出版社,2008.545-581.Ausubel F.Short Protocols in Molecular Biology.Fifth ed.Beijing:Science Press,2008.545-581.
[25]Zhang B,Rong C,Chen H,et al.De novo synthesis of trans-10,cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia lipolytica.Microb Cell Fact,2012,11(1):51.