适于烟草生物反应器的真核生物源虾青素合成酶筛选
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摘要
为揭示不同真核生物源虾青素合成酶在烟草中的异源表达生物活性特征,明确开发虾青素烟草生物反应器的关键分子基础,本研究对夏侧金盏花(Adonis aestivalis)、雨生红球藻(Haematococcus pluvialis)及红发夫酵母(Xanthophyllomyces dendrorhous)3种真核生物源虾青素合成酶基因进行了密码子优化合成及表达载体构建,通过农杆菌介导的瞬时表达和稳定表达试验比较了其在烟草中合成虾青素的活性差异。结果表明,3种来源的虾青素合成酶基因均能在烟草中表达,但是仅夏侧金盏花和雨生红球藻来源的合成酶能在烟草中特异合成虾青素,且夏侧金盏花虾青素合成酶的活性最高。此外,稳定表达体系优于瞬时表达体系,能准确展示不同虾青素合成酶的活性差异。本研究为真核生物源虾青素合成酶在烟草生物反应器中的应用提供了重要基础。
In order to determine the bioactivity of eukaryotes astaxanthin synthase ectopically expressed in tobacco, and to reveal the molecular basis for the development of tobacco bioreactor for astaxanthin, we codon-optimized and synthesized three eukaryotic-type astaxanthin synthases from Adonis aestivalis, Haematococcus pluvialis and Xanthophyllomyces dendrorhous respectively, and compared their astaxanthin synthetic bioactivities in tobacco using both transient and stable expression experiments.The results showed that the genes of the three resources were all successfully expressed in tobacco, whereas only the A. aestivalisand H. pluvialis-derived synthases could specifically synthesize astaxanthin, and the A. aestivalis-derived astaxanthin synthases possessed a higher activity. The results also suggested that the stable expression system was more sensitive than the transient expression system in determining the activity of astaxanthin synthetic enzymes in tobacco. This study has provided molecular basis for the construction of tobacco bioreactor for astaxanthin synthesis.
In order to determine the bioactivity of eukaryotes astaxanthin synthase ectopically expressed in tobacco, and to reveal the molecular basis for the development of tobacco bioreactor for astaxanthin, we codon-optimized and synthesized three eukaryotic-type astaxanthin synthases from Adonis aestivalis, Haematococcus pluvialis and Xanthophyllomyces dendrorhous respectively, and compared their astaxanthin synthetic bioactivities in tobacco using both transient and stable expression experiments.The results showed that the genes of the three resources were all successfully expressed in tobacco, whereas only the A. aestivalisand H. pluvialis-derived synthases could specifically synthesize astaxanthin, and the A. aestivalis-derived astaxanthin synthases possessed a higher activity. The results also suggested that the stable expression system was more sensitive than the transient expression system in determining the activity of astaxanthin synthetic enzymes in tobacco. This study has provided molecular basis for the construction of tobacco bioreactor for astaxanthin synthesis.
引文
[1]HIGUERA-CIAPARA I,FELIX-VALENZUELA L,GOYCOOLEA F M.Astaxanthin:a review of its chemistry and applications[J].Critical Reviews in Food Science and Nutrition,2006,46(2):185-196.
[2]JOHNSON E A,AN G-H.Astaxanthin from microbial sources[J].Critical Reviews in Biotechnology,1991,11(4):297-326.
[3]HUSSEIN G,SANKAWA U,GOTO H,et al.Astaxanthin,a carotenoid with potential in human health and nutrition[J].Journal of Natural Products,2006,69(3):443-449.
[4]GUERIN M,HUNTLEY M E,OLAIZOLA M.Haematococcus astaxanthin:applications for human health and nutrition[J].Trends in Biotechnology,2003,21(5):210-216.
[5]SHAH M M R,LIANG Y,CHENG J J,et al.Astaxanthin-poducing preen microalga Haematococcus pluvialis:from single cell to high value commercial products[J].Frontiers in Plant Science,2016,7:531.
[6]KOLLER M,MUHR A,BRAUNEGG G.Microalgae as versatile cellular factories for valued products[J].Algal Research,2014,6:52-63.
[7]PéREZ-LóPEZ P,GONZáLEZ-GARCíA S,JEFFRYES C,et al.Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae:from lab to pilot scale[J].Journal of Cleaner Production,2014,64(64):332-344.
[8]TREMBLAY R,WANG D,JEVNIKAR A M,et al.Tobacco,a highly efficient green bioreactor for production of therapeutic proteins[J].Biotechnology Advances,2010,28(2):214-221.
[9]时焦,J I COOPER.几丁质酶基因在烟草栽培品种中的表达[J].中国烟草科学,1997,18(4):47-50.SHI J,COOPER J I.Expression of chitinase gene in tobacco cultivars[J].Chinese Tobacco Science,1997,18(4):47-50.
[10]李雪君,崔红,刘海礁,等.Fbs转基因烤烟类胡萝卜素及其降解产物的研究[J].中国烟草科学,2006,27(3):25-27.LI X J,CUI H,LIU H J,et al.Study on carotenoids and their degradation products of Fbs transgenic flue-cured tobacco[J].Chinese Tobacco Science,2006,27(3):25-27.
[11]MANN V,HARKER M,PECKER I,et al.Metabolic engineering of astaxanthin production in tobacco flowers[J].Nature Biotechnology,2000,18(8):888-892.
[12]HASUNUMA T,MIYAZAWA S,YOSHIMURA S,et al.Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering[J].The Plant Journal,2008,55(5):857-868.
[13]MISAWA N,SATOMI Y,KONDO K,et al.Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level[J].Journal of Bacteriology,1995,177(22):6575-6584.
[14]DUCREY SANPIETRO L M,KULA M-R.Studies of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous(Phaffia rhodozyma).Effect of inhibitors and low temperature[J].Yeast,1998,14(11):1007-1016.
[15]LEE J H,KIM Y T.Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis[J].Gene,2006,370:86-95.
[16]YOKOYAMA A,MIKI W.Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin‐producing bacterium Agrobacterium aurantiacum[J].FEMS Microbiology Letters,1995,128(2):139-144.
[17]KAJIWARA S,KAKIZONO T,SAITO T,et al.Isolation and functional identification of a novel c DNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli[J].Plant Molecular Biology,1995,29(2):343-352.
[18]CUNNINGHAM F X,GANTT E.A study in scarlet:enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis[J].The Plant Journal,2005,41(3):478-492.
[19]CUNNINGHAM F X,GANTT E.Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis[J].The Plant Cell,2011,23(8):3055-3069.
[20]OJIMA K,BREITENBACH J,VISSER H,et al.Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous(Phaffia rhodozyma)and its assignment as aβ-carotene 3-hydroxylase/4-ketolase[J].Molecular Genetics&Genomics,2006,275(2):148-158.
[21]MARTíN J F.GUDI?A E,BARREDO J L.Conversion ofβ-carotene into astaxanthin:Two separate enzymes or a bifunctional hydroxylase-ketolase protein?[J].Microbial Cell Factories.,2008,7(1):3.
[22]高玉龙,肖炳光,Ralph E DEWEY.诱导烟草早花的PVX-FT系统的建立[J].中国烟草学报,2013,19(6):102-105.GAO Y L,XIAO B G,DEWEY R E.Construction of PVX-FT system that inducing early flowering in tobacco[J].Acta Tabacaria Sinica,2013,19(6):102-105.
[23]NAKRIEKO K-A,MOULD R M,SMITH A G.Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide[J].European Journal of Biochemistry,2004,271(3):509-516.
[2]JOHNSON E A,AN G-H.Astaxanthin from microbial sources[J].Critical Reviews in Biotechnology,1991,11(4):297-326.
[3]HUSSEIN G,SANKAWA U,GOTO H,et al.Astaxanthin,a carotenoid with potential in human health and nutrition[J].Journal of Natural Products,2006,69(3):443-449.
[4]GUERIN M,HUNTLEY M E,OLAIZOLA M.Haematococcus astaxanthin:applications for human health and nutrition[J].Trends in Biotechnology,2003,21(5):210-216.
[5]SHAH M M R,LIANG Y,CHENG J J,et al.Astaxanthin-poducing preen microalga Haematococcus pluvialis:from single cell to high value commercial products[J].Frontiers in Plant Science,2016,7:531.
[6]KOLLER M,MUHR A,BRAUNEGG G.Microalgae as versatile cellular factories for valued products[J].Algal Research,2014,6:52-63.
[7]PéREZ-LóPEZ P,GONZáLEZ-GARCíA S,JEFFRYES C,et al.Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae:from lab to pilot scale[J].Journal of Cleaner Production,2014,64(64):332-344.
[8]TREMBLAY R,WANG D,JEVNIKAR A M,et al.Tobacco,a highly efficient green bioreactor for production of therapeutic proteins[J].Biotechnology Advances,2010,28(2):214-221.
[9]时焦,J I COOPER.几丁质酶基因在烟草栽培品种中的表达[J].中国烟草科学,1997,18(4):47-50.SHI J,COOPER J I.Expression of chitinase gene in tobacco cultivars[J].Chinese Tobacco Science,1997,18(4):47-50.
[10]李雪君,崔红,刘海礁,等.Fbs转基因烤烟类胡萝卜素及其降解产物的研究[J].中国烟草科学,2006,27(3):25-27.LI X J,CUI H,LIU H J,et al.Study on carotenoids and their degradation products of Fbs transgenic flue-cured tobacco[J].Chinese Tobacco Science,2006,27(3):25-27.
[11]MANN V,HARKER M,PECKER I,et al.Metabolic engineering of astaxanthin production in tobacco flowers[J].Nature Biotechnology,2000,18(8):888-892.
[12]HASUNUMA T,MIYAZAWA S,YOSHIMURA S,et al.Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering[J].The Plant Journal,2008,55(5):857-868.
[13]MISAWA N,SATOMI Y,KONDO K,et al.Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level[J].Journal of Bacteriology,1995,177(22):6575-6584.
[14]DUCREY SANPIETRO L M,KULA M-R.Studies of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous(Phaffia rhodozyma).Effect of inhibitors and low temperature[J].Yeast,1998,14(11):1007-1016.
[15]LEE J H,KIM Y T.Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis[J].Gene,2006,370:86-95.
[16]YOKOYAMA A,MIKI W.Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin‐producing bacterium Agrobacterium aurantiacum[J].FEMS Microbiology Letters,1995,128(2):139-144.
[17]KAJIWARA S,KAKIZONO T,SAITO T,et al.Isolation and functional identification of a novel c DNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli[J].Plant Molecular Biology,1995,29(2):343-352.
[18]CUNNINGHAM F X,GANTT E.A study in scarlet:enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis[J].The Plant Journal,2005,41(3):478-492.
[19]CUNNINGHAM F X,GANTT E.Elucidation of the pathway to astaxanthin in the flowers of Adonis aestivalis[J].The Plant Cell,2011,23(8):3055-3069.
[20]OJIMA K,BREITENBACH J,VISSER H,et al.Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous(Phaffia rhodozyma)and its assignment as aβ-carotene 3-hydroxylase/4-ketolase[J].Molecular Genetics&Genomics,2006,275(2):148-158.
[21]MARTíN J F.GUDI?A E,BARREDO J L.Conversion ofβ-carotene into astaxanthin:Two separate enzymes or a bifunctional hydroxylase-ketolase protein?[J].Microbial Cell Factories.,2008,7(1):3.
[22]高玉龙,肖炳光,Ralph E DEWEY.诱导烟草早花的PVX-FT系统的建立[J].中国烟草学报,2013,19(6):102-105.GAO Y L,XIAO B G,DEWEY R E.Construction of PVX-FT system that inducing early flowering in tobacco[J].Acta Tabacaria Sinica,2013,19(6):102-105.
[23]NAKRIEKO K-A,MOULD R M,SMITH A G.Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide[J].European Journal of Biochemistry,2004,271(3):509-516.